Screening method

ABSTRACT

The screening method for a compound or a salt thereof that alters the binding property between Neuromedin U or a salt thereof and TGR-1 or a salt thereof, characterized by using Neuromedin U, a derivative thereof or a salt thereof and TGR-1 or a salt thereof, can be useful for screening a therapeutic and/or prophylactic agent for hypertension and stress-related diseases, etc. A TGR-1 antagonist can be useful as a therapeutic and/or prophylactic agent for hypertension and stress-related diseases, etc.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel receptor protein TGR-1derived from human testis and rat uterus, a DNA encoding TGR-1, and ascreening method for a prophylactic and/or therapeutic agent forhypertension or stress-related diseases, or an agent for controllingappetite, which is characterized by using TGR-1 and Neuromedin U(Minamino, N. et al., Biochem. Biophys. Res. Commmun. 130, 1078-1085,1985), or derivatives or salts thereof.

BACKGROUND ART

[0002] A variety of hormones and neurotransmitters regulate thebiological functions through specific receptor proteins located in acell membrane. Many of these receptor proteins are coupled with guaninenucleotide-binding proteins (hereinafter sometimes referred to as Gproteins) and evoke the intracellular signal transduction via activationof the G proteins. These receptor proteins possess the common structure,i.e. seven transmembrane domains and are thus collectively referred toas G protein-coupled receptors or seven-transmembrane receptors (7TMR).

[0003] An important regulation of biological functions, such ashomeostasis, reproduction, individual development, metabolism, growth,regulations of nervous system, respiratory system, digestive system andmetabolic system, and sensory system is conducted through an interactionbetween these hormones or neurotransmitters and G protein-coupledreceptor proteins. In this context, it is known that there are variousreceptor proteins for hormones and neurotransmitters for the regulationof biological functions and these proteins play an important role forregulating the functions. However, it is not much clear as to whetherunknown active substances (e.g. hormones, neurotransmitters, etc.) andreceptors thereof still exist.

[0004] In recent years, using the fact that G protein-coupled receptorproteins represent similarities in their partial amino acid sequences,the search for DNA encoding a novel receptor protein is conducted byPolymerase Chain Reaction (hereinafter abbreviated as PCR) method.Therefore, many orphan G protein-coupled receptor proteins whose ligandare not known, are cloned (Libert, F., et al. Science, 244, 569-572,1989, Welch, S. K., et al., Biochem. Biophys. Res. Commun., 209,609-613, 1995, Marchese, A., et al., Genomics, 23, 609-618, 1994,Marchese, A., Genomics, 29, 335-344, 1995). Novel G protein-coupledreceptor proteins are also found by random analysis of genomic DNA orcDNA sequences (Nomura, N., et al., DNA Research vol. 1, 27-25,1994).General methods for determining a ligand to an orphan G protein-coupledreceptor protein are only to predict the ligand from similarity of theprimary structure of G protein-coupled receptor protein. However, sincemany G protein-coupled receptor proteins represent low homology with theknown receptors, it is difficult to predict a ligand only from thesimilarity of the primary structure unless the receptor protein is asubtype receptor for the known ligand. On the other hand, many orphan Gprotein-coupled receptor proteins are found by genetic analysis. So, itis estimated that there are many unknown ligands still remained.Nevertheless, only a few ligands for G protein-coupled receptor proteinsare actually identified.

[0005] On the other hand, Neuromedin U is a peptide, which was isolatedand purified from porcine spinal cords, with setting a rat uterus smoothmuscle contraction activity as an index. Two kinds of Neuromedin U,Neuromedin U-8 having 8 amino acid residues and Neuromedin U-25 having25 amino acid residues are first reported (Minamino, N. et al., Biochem.Biophys. Res. Commun. 130, 1078-1085, 1985). Since the sequence ofNeuromedin U-8 is identical to C-terminal sequence of Neuromedin U-25and the upstream region contains a basic amino acid pair often seen inthe cleavage site for processing, both Neuromedin U are expected to bederived from a common precursor. Also, other physiological functionsbesides the smooth muscle contraction activity are widely known. Suchfunctions reportedly include, for example, increase in blood pressure(Minamino. N. et al.), decrease in bloodstream of intestine (Sumi, S. etal., Life Sci. 41, 1585-1590, 1987), adjustment of ion transportation inintestine (Brown, D. R. and Quito, F. L., Eur. J. Pharmacol. 155,159-162, 1998) and increase in ACTH and subsequent increase incorticosterone after hypodermic administration of Neuromedin U(Malendowicz, L. K. et al., In Vivo, 7, 419-422, 1993).

SUMMARY OF THE INVENTION

[0006] Until now, only FM-3 has been identified as a receptor forNeuromedin U (WO 00/02918). However, there is a need to develop a newdrug by finding a receptor for Neuromedin U other than FM-3, clarifingthe physiological role of Neuromedin U, and screening a compound thatactivates or inhibits its action.

[0007] The present inventors have found, with extensive investigation, anew orphan G protein-coupled receptor protein TGR-1, and also foundunexpectedly that Neuromedin U has the cell stimulating activity to theTGR-1-expressing CHO cells in a specific manner. Based on thesefindings, the present inventors continued extensive studies toaccomplish the present invention.

[0008] Thus, the present invention relates to the followings:

[0009] (1) A method for screening a compound or a salt thereof thatalters the binding property of Neuromedin U or a salt thereof with aprotein or a salt thereof comprising the same or substantially the sameamino acid sequence as the sequence shown by SEQ ID NO:1 or NO:21, whichis characterized by using Neuromedin U, a derivative thereof or a saltthereof and a protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.

[0010] (2) A kit for screening a compound or a salt thereof that altersthe binding property of Neuromedin U or a salt thereof with a protein ora salt thereof comprising the same or substantially the same amino acidsequence as the sequence shown by SEQ ID NO:1 or NO:21, which ischaracterized by comprising Neuromedin U, a derivative thereof or a saltthereof and a protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.

[0011] (3) A compound or a salt thereof that alters the binding propertyof Neuromedin U or a salt thereof with a protein or a salt thereofcomprising the same or substantially the same amino acid sequence as thesequence shown by SEQ ID NO:1 or NO:21, which is obtainable using thescreening method described in (1) or the screening kit described in (2).

[0012] (4) A pharmaceutical composition comprising the compounddescribed in (3).

[0013] (5) A pharmaceutical composition described in (4), which is atherapeutic and prophylactic agent for obesity, hypertension orstress-related diseases.

[0014] (6) A screening method described in (1) or a screening kitdescribed in (2), where Neuromedin U is a peptide comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:11.

[0015] (7) A protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.

[0016] (8) A DNA containing the DNA encoding the protein described in(7).

[0017] (9) A DNA described in (8) having the base sequence shown by SEQID NO:2 or No:22.

[0018] (10) A recombinant vector containing the DNA described in (8).

[0019] (11) A transformant transformed with the recombinant vectordescribed in (10).

[0020] (12) A method for producing the protein or salt thereof describedin (7), characterized by cultivating the transformant described in (11)and making the transformant produce the protein described in (7).

[0021] (13) An antibody to the protein or salt thereof described in (7).

[0022] In the present invention, Neuromedin U specifically includes theabove-mentioned Neuromedin U or a salt thereof, and also:

[0023] (14) Neuromedin U includes proteins (polypeptides) comprising theamino acid sequence shown by SEQ ID NO:5, NO:6, NO:7, NO:8, NO:9, NO:10,NO:12, NO:13, NO:14 or NO:15, or derivatives or salts thereof.

[0024] For Neuromedin U of the present invention, it is preferred thatthe carboxyl group of the C-terminal amino acid is amidated.

[0025] TGR-1 of the present invention specifically includes the proteinor a salt thereof comprising the same or substantially the same aminoacid sequence as the sequence shown by SEQ ID NO:1 or NO:21, and also:

[0026] (15) TGR-1 includes the protein or a salt thereof comprising thesame or substantially the same amino acid sequence as the sequence shownby SEQ ID NO:17;

[0027] (16) TGR-1 includes proteins or salts thereof comprising theamino acid sequence shown by SEQ ID NO:1, NO:17 or NO:21 wherein 1 to 30amino acids, preferably 1 to 10 amino acids are deleted, wherein 1 to 30amino acids, preferably 1 to 10 amino acids are added (iserted), wehrein1 to 30 amino acids, preferably 1 to 10 amino acids are substituted withother amino acids.

[0028] More specifically, TGR-1 includes proteins comprising the partfrom 4 (Met) through 415 (Thr) positions of the amino acid sequenceshown by SEQ ID NO:1 or NO:17.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows the result of detection of TGR-1 receptor-specificcell-stimulating activity using the site sensor, performed in Example 2.In the site sensor assay, pig Neuromedin U-8 at the concentrationsindicated in FIG. 1 was reacted to TGR-1-expressing CHO cells () andmock CHO cells (◯) for 7 minutes and 2 seconds. The maximum ofacidification rate during the reaction was plotted.

[0030]FIG. 2 shows the comparison of human TGR-1 obtained in Example 1and rat TGR-1 obtained in Example 3 in terms of amino acid sequence.

BEST MODE OF THE INVENTION

[0031] The details of method for producing TGR-1 or a salt thereof(hereinafter simply referred to as TGR-1) and Neuromedin U, a derivativethereof or a salt thereof (hereinafter simply referred to as NeuromedinU) are described as follows.

[0032] TGR-1 and Neuromedin U of the present invention may be anyproteins ((poly)peptides) derived from any tissues (e.g. hypophysis,pancreas, brain, kidney, liver, gonad, thyroid, gallbladder, bonemarrow, adrenal gland, skin, muscle, lung, gastrointestinal tract, bloodvessel, heart, etc.) or any cells from warm-blooded animals (e.g. human,guinea pig, rat, mouse, swine, sheep, bovine, monkey, dog, chicken),amphibian (e.g. frog) and fish. TGR-1 may be any proteins((poly)peptides) having the same or substantially the same amino acidsequence as that shown by SEQ ID NO:1 or NO:21, and Neuromedin U may beany proteins having the same or substantially the same amino acidsequence as that shown by SEQ ID NO:11.

[0033] TGR-1 includes a protein having the substantially same activityas that of the protein having the amino acid sequence shown by SEQ IDNO:1 or NO:21, as well as the protein containing the amino acid sequenceshown by SEQ ID NO:1 or NO:21.

[0034] Herein, the term “substantially same” means the substantialequiavalence in the binding activity of a ligand (Neuromedin U) and areceptor (TGR-1), a physiological property or the like. Thesubstitution, deletion, addition and insertion of amino acids in aplypeptide often do not give a detectable change in physiological andchemical properties of the polypeptide. In such case, the protein((poly)peptide) that is modified by the substitution, deletion, additionor insertion (so called a variant of Neuromedin U, TGR-1, or the like)is considered to be substantially the same as the protein which is notmodidifed.

[0035] The amino acid in said amino acid sequence can be substitutedwith substantially the same amino acid selected from, for example, otheramino acids of the group that the amino acid belongs to. Non-polar(hydrophobic) amino acid includes alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, methionine, etc. Polar (neutral)amino acid includes glycine, serine, threonin, cycteine, thyrosine,asparagine, glutamine, etc. Positively charged (basic) amino acidincludes arginine, lysine, histidine, etc. Negatively charged (acidic)amino acid includes aspartic acid, glutamic acid, etc.

[0036] Therefore, these proteins may vary in quantitative factors suchas a binding activity level, a molecular weight, etc.

[0037] More specific examples of the substantially same amino acidsequence as the sequence shown by SEQ ID NO:1 or NO:21 include an aminoacid sequence with at least about 90%, more preferably at least about95%, even more preferably at least about 98% homology with the sequenceshown by SEQ ID NO:1 or NO:21.

[0038] In particular, examples of the substantially same amino acidsequence as the sequence shown by SEQ ID NO:1 include an amino acidsequence which comprises the amino acid sequence of Leu-Phe-Val,Trp-Ser-Glu, Val-Phe-Phe, or Ser-Met-His as a partial sequence, and haspreferably at least about 90%, more preferably at least about 95%, evenmore preferably at least about 98% homology with the sequence shown bySEQ ID NO:1.

[0039] Preferred examples of a protein comprising the substantially sameamino acid sequence as the sequence shown by SEQ ID NO:1 or NO:21include a protein which has the substantially same amino acid sequenceas the sequence shown by SEQ ID NO:1 or NO:21, and the substantiallysame activity as that of the amino acid sequence shown by SEQ ID NO:1 orNO:21.

[0040] Examples of a protein comprising the substantially same aminoacid sequence as the sequence shown by SEQ ID NO:1 include a proteinswhich comprises the amino acid sequence of Leu-Phe-Val, Trp-Ser-Glu,Val-Phe-Phe, or Ser-Met-His as a partial sequence, or preferably has thesubstantially same amino acid sequence as the sequence shown by SEQ IDNO:1, and the substantially same activity as that of the amino acidsequence shown by SEQ ID NO:1.

[0041] Examples of “the substantially same activity” include the ligandbinding activity, the signal transducing activity, etc. The term“substantially same” means the qualitative equivalence in theseactivities. Thus, it is preferred that an activity, such as the ligandbinding activity, the signal transducing activity, etc. isquantitatively equivalent (for example, about 0.01 to 100 times,preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times),but quantitative factors, such as levels of these activity, a molecularweight, etc. may vary.

[0042] These activities, such as the ligand binding activity, the signaltransducing activity can be measured according to a known method, forexample, the screening method as described below.

[0043] In addition, examples of the substantially same amino acidsequence as the sequence shown by SEQ ID NO:1 or NO:21 include (i) anamino acid sequence in which one or more (preferably 1 to 30, morepreferably 1 to 10, and even more preferably several (1 to 5)) aminoacids are deleted from the amino acid sequence represented by SEQ IDNO:1 or NO:21; (ii) an amino acid sequence in which one or more(preferably 1 to 30, more preferably 1 to 10, and even more preferablyseveral (1 to 5)) amino acids are added to the amino acid sequencerepresented by SEQ ID NO:1 or NO:21; (iii) an amino acid sequence inwhich one or more (preferably 1 to 30, more preferably 1 to 10, and evenmore preferably several (1 to 5)) amino acids are substituted with otheramino acids in the amino acid sequence represented by SEQ ID NO:1 orNO:21; and (iv) an amino acid sequence comprising any combination of theabove modifications.

[0044] In addition, examples of the substantially same amino acidsequence as the sequence shown by SEQ ID NO:1 include (i) an amino acidsequence in which one or more (preferably 1 to 30, more preferably 1 to10, and even more preferably several (1 to 5)) amino acids are deletedfrom the amino acid sequence represented by SEQ ID No:1, and whichcomprises the amino acid sequence of Leu-Phe-Val, Trp-Ser-Glu,Val-Phe-Phe, or Ser-Met-His as a partial sequence; (ii) an amino acidsequence in which one or more (preferably 1 to 30, more preferably 1 to10, and even more preferably several (1 to 5)) amino acids are added tothe amino acid sequence represented by SEQ ID NO:1, and which comprisesthe amino acid sequence of Leu-Phe-Val, Trp-Ser-Glu, Val-Phe-Phe, orSer-Met-His as a partial sequence; (iii) an amino acid sequence in whichone or more (preferably 1 to 30, more preferably 1 to 10, and even morepreferably several (1 to 5)) amino acids are substituted with otheramino acids in the amino acid sequence represented by SEQ ID NO:1, andwhich comprises the amino acid sequence of Leu-Phe-Val, Trp-Ser-Glu,Val-Phe-Phe, or Ser-Met-His as a partial sequence; and (iv) an aminoacid sequence comprising any combination of the above modifications.

[0045] Furthermore, examples of TGR-1 include a protein comprising aportion from 4th (Met) to 415th (Thr) position from the N-terminal ofthe amino acid sequence shown by SEQ ID NO:1 or NO:17.

[0046] On the other hand, examples of Neuromedin U of the presentinvention include (poly)peptides having the substantially same activityas that of the (poly)peptide having the amino acid sequence shown by SEQID NO:11, in addition to the (poly)peptide having the amino acidsequence shown by SEQ ID NO:11.

[0047] Examples of “the substantially same activity” include the bindingactivity of the receptor, and the like. The term “substantially same”means the qualitative equivalence, for example, in the binding activityof the receptor, and the like. Therefore, quantitative factors such as abinding activity level, a molecular weight, etc. may vary.

[0048] In the present specification, the amino acid sequences of TGR-1and Neuromedin U are shown so that the N-terminal (amino terminal) isplaced in the left and the C-terminal (carboxyl terminal) in the right,in accordance with a conventional peptide notation system. The proteinor (poly)peptide having the amino acid sequence shown by SEQ ID NO: 1 orNO:21, or NO:11 usually has a carboxyl group (—COOH) or carboxylate(—COO⁻) at the C-terminal, but may have an amide (—CONH₂) or ester(—COOR) at the C-terminal.

[0049] R in said ester includes, for example, C₁₋₆ alkyl groups such asmethyl, ethyl, n-propyl, isopropyl and n-butyl; C₃₋₈ cycloalkyl groupssuch as cyclopentyl and cyclohexyl; C₆₋₁₂ aryl groups such as phenyl andα-naphthyl; C₇₋₁₄ aralkyl groups such as phenyl-C₁₋₂ alkyl, such asbenzyl, phenethyl and benzhydryl, and α-naphthyl-C₁₋₂ alkyl, such asα-naphthylmethyl; and pivaloyloxymethyl groups generally used as anester suitable for oral administration.

[0050] Preferred example of Neuromedin U of the present invention is theone having an amide (—CONH₂) at the C-terminal.

[0051] Examples of salts of TGR-1 or Neuromedin U used in the presentinvention include salts with physiologically acceptable bases (e.g.,alkali metals) or acids (e.g., inorganic acids, organic acids).Specially, physiologically acceptable acid addition salts are preferred.Such salts include, for example, salts with inorganic acids (e.g.,hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) orsalts with organic acids (e.g., acetic acid, formic acid, propionicacid, fumaric acid, maleic acid, succinic acid, tartaric acid, citricacid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,benzenesulfonic acid).

[0052] The TGR-1 or Neuromedin U used in the present invention can beproduced by a known method (the method described in FEBS Letters,398(1996), 253-258, or WO 96/18651). That is, it can be produced by apolypeptide purification technique from the human or warm-blooded animalcells or tissues, or can be produced according to a peptide synthesismethod described below. Alternatively, it can be produced by culturing atransformant containing the DNA encoding the protein (peptide) describedbelow.

[0053] When the protein is produced from tissues or cells of human,warm-blooded animal, amphibian or fish, the tissues or cells arehomogenized, then extracted with an acid, an organic solvent or thelike, and then the protein is isolated and purified from the extract bya combination of chromatography techniques such as salting out,dialysis, gel filtration, reverse-phase chromatography, ion-exchangechromatography, affinity chromatography, etc.

[0054] TGR-1 or Neuromedin U used in the present invention can beproduced according to a known method for peptide synthesis or bycleaving a protein ((poly)peptide) containing TGR-1 or Neuromedin U witha suitable peptidase. For example, the protein ((poly)peptide) synthesismethod may be the solid- or liquid-phase synthesis method. That is, thedesired protein ((poly)peptide) can be obtained by condensation ofpartial peptides or amino acids composing TGR-1 or Neuromedin U with theremaining parts, followed by elimination of protecting groups, if any,from the product.

[0055] The known methods for condensation and elimination of protectinggroups can be found in e.g. the following (1) to (5):

[0056] (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis,Interscience Publisher, New York (1966);

[0057] (2) Schroeder and Luebke, The Peptide, Academic Press, New York(1965);

[0058] (3) Nobuo Izumiya et al., Basis and Experiments in PeptideSynthesis, Maruzen Co., Ltd. (1975);

[0059] (4) Haruaki Yajima and Shunpei Sakakibara, BiochemicalExperimental Course 1, Protein Chemistry IV, 205, (1977); and

[0060] (5) Haruaki Yajima (supervisor), Development of medicines, asecond series, vol.14, Peptide Synthesis, Hirokawashoten.

[0061] After the reaction, the protein ((poly)peptide) can be isolatedand purified by a combination of conventional purification techniquessuch as solvent extraction, distillation, column chromatography, liquidchromatography and recrystallization. If the protein ((poly)peptide) isobtained in a free form by these methods, the product can be convertedinto a suitable salt by a known method, or if the protein((poly)peptide) is obtained in a salt form, it can be converted into afree form by a known method.

[0062] For synthesis of amide derivative of TGR-1 or Neuromedin U,usually commercially available resin for protein synthesis can be used.Such resin includes, for example, chloromethyl resin, hydroxymethylresin, benzhydryl amine resin, aminomethyl resin, 4-benzyloxybenzylalcohol resin, 4-methylbenzhydryl amine resin, PAM resin,4-hydroxymethylmethylphenylacetamidemethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc aminoethyl)phenoxy resin, and so forth. Onthe resin described above, each amino acid with the α-amino group andside-chain functional group properly protected is condensed sequentiallyin accordance with the sequence of the desired peptide by the per seknown condensation methods. At the end of the reaction, the protein((poly)peptide) is cleaved off from the resin, and various protectinggroups are removed, and the product is subjected to a reaction offorming intramolecular disulfide bonds in a highly dilute solution togive the desired protein ((poly)peptide).

[0063] A wide variety of activating reagents usable for proteinsynthesis can be used for condensation of the protected amino acidsdescribed above, and carbodiimides are particularly preferable. Examplesof such carbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids along with racemizationinhibitors (e.g., HOBt, HOOBt) can be added to the resin directly orafter the protected amino acids were previously activated as symmetricacid anhydrides or HOBt esters or HOOBt esters. The solvent used foractivation of each protected amino acid or for condensation thereof withthe resin can be selected as necessary from those solvents known to beusable in protein ((poly)peptide) condensation reaction. Examples ofsuch solvent include acid amides such as N,N-dimethylformamide,N,N-dimethylacetamide and N-methylpyrrolidone; halogenated hydrocarbonssuch as methylene chloride and chloroform; alcohols such astrifluoroethanol; sulfoxides such as dimethyl sulfoxide; tertiary aminessuch as pyridine; ethers such as dioxane and tetrahydrofuran; nitritessuch as acetonitrile and propionitrile; esters such as methyl acetateand ethyl acetate, or a suitable mixture thereof. The reactiontemperature is usually selected as necessary within the range known tobe usable in the reaction of forming peptide bonds, and usually thereaction temperature is selected within the range of about −20° C. to50° C. The activated amino acid derivatives are used usually in excess(1.5- to 4-fold). When their condensation is insufficient as a result ofa ninhydrin reaction test, their sufficient condensation is achieved byrepeatedly carrying out the condensation reaction without conductingelimination of the protecting groups. When their sufficient condensationis not achieved even by repeatedly carrying out the reaction, theunreacted amino acids are acetylated with acetic anhydride or acetylimidazole so that the subsequent reaction cannot be influenced.

[0064] The protecting groups for amino groups in amino acids as thestarting materials include, for example, Z, Boc, t-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosfinothioyl, Fmoc etc.

[0065] The carboxyl group can be protected by, for example, C₁₋₆ alkyl,C₃₋₈ cycloalkyl, C₇₋₁₄ aralkyl as above described for, or 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazide, t-butoxycarbonylhydrazide, tritylhydrazide,etc.

[0066] The hydroxyl group in serine and threonine can be protected by,for example, esterification or etherification. A suitable group used inthis esterification includes, for example, lower alkanoyl groups such asacetyl group; alloyl groups such as benzoyl group; and carbonicacid-derived groups such as benzyloxycarbonyl group and ethoxycarbonylgroup. A suitable group for etherification includes, for example, abenzyl group, tetrahydropyranyl group, t-butyl group, etc.

[0067] The protecting group used for the phenolic hydroxyl group intyrosine includes, for example, Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z,t-butyl etc.

[0068] The protecting group used for imidazole in histidine includes,for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc etc.

[0069] Activated carboxyl groups in the starting materials include, forexample, the corresponding acid anhydrides, azides and active esters(i.e. esters with alcohols such as pentachlorophenol,2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol,p-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide andHOBt). The activated amino groups in the starting materials include, forexample, the corresponding phosphoric acid amides.

[0070] Examples of methods for removing (leaving) of the protectinggroups include catalytic reduction in a hydrogen stream in the presenceof a catalyst such as Pd-black or Pd-carbon; acid treatment usinganhydrous hydrogen fluoride, methane sulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixed solution thereof; basetreatment using diisopropylethylamine, triethylamine, piperidine orpiperazine; and reduction using sodium in liquid ammonia. The leavingreaction by the acid treatment is carried out generally at a temperatureof about −20° C. to 40° C., and it is useful in the acid treatment toadd a cation scavenger such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethylsulfide, 1,4-butanedithiol and 1,2-ethanedithiol. A2,4-dinitrophenyl group used as a protecting group for imidazole inhistidine can also be removed by treatment with thiophenol, while aformyl group used as a protecting group for indole in tryptophan can beremoved for deprotection by acid treatment in the presence of1,2-ethanedithiol or 1,4-butanedithiol above, or also by alkalitreatment using dilute sodium hydroxide solution or dilute ammonia.

[0071] Protection and protecting groups for functional groups whichshould not participate in the reaction of the starting materials,elimination of the protecting groups, and activation of functionalgroups participating in the reaction can be selected as necessary fromknown groups or known means.

[0072] Another method of obtaining an amide derivative of TGR-1 orNeuromedin U includes, for example, amidating the α-carboxyl group of aC-terminal amino acid, then extending a peptide chain at the side of theamino group until it attains desired chain length, and thereafterproducing a peptide of said peptide chain from which only the protectinggroup for the N-terminal α-amino group was removed and a peptide of saidpeptide chain from which only the protecting group for the C-terminalcarboxyl group was removed, followed by condensation both the proteinsin the mixed solvent as described above. The details of the condensationreaction are the same as described above. The protected protein obtainedby condensation is purified, and every protecting group is removed bythe method descried above, whereby the desired crude protein((poly)peptide) can be obtained. This crude protein ((poly)peptide) ispurified by a wide variety of known purification techniques, and bylyophilizing its major fraction, the desired amide derivative of theprotein ((poly)peptide) can be obtained.

[0073] To obtain an ester derivative of TGR-1 or Neuromedin U, forexample, the α-carboxyl group of a C-terminal amino acid is condensedwith desired alcohol to form an amino acid ester, from which the desiredester derivative of the protein ((poly)peptide) can be obtained in thesame manner as for the amide derivative of the protein ((poly)peptide).

[0074] The Neuromedin U derivatives used in the present invention may beany having a binding activity with TGR-1, such as (1) a partial peptideof Neuromedin U; (2) a peptide wherein the constitutive amino acid ofNeuromedin U was deleted, a peptide wherein other amino acids is addedto the constitutive amino acid, a peptide wherein the constitutive aminoacid is substituted by other amino acids; or (3) a labeled Neuromedin U,a labeled partial peptide described in (1) or a labeled peptidedescribed in (2).

[0075] Specifically, the partial peptide of Neuromedin U includes thepeptide having the amino acid sequence shown by SEQ ID NO:16, its amidederivatives, its ester derivatives or a salt thereof. Among those, theamide derivatives of the peptide having the amino acid sequence shown bySEQ ID NO:16 or its salt are preferred.

[0076] The partial peptide of Neuromedin U can be produced by cleavingNeuromedin U desclosed in the above with a suitable peptidase oraccording to the above-mentioned protein ((poly)peptide) synthesismethod. The amide derivatives and the ester derivatives of the partialpeptide of Neuromedin U can be produced according to the above-mentionedamide derivative production method or the above-mentioned esterderivative production method. Moreover, salt of the partial peptide ofNeuromedin U includes the same salts as the above-mentioned salt ofTGR-1 or Neuromedin U.

[0077] Examples of the Neuromedin U peptide wherein any constitutiveamino acids of Neuromedin U are deleted, wherein other amino acids areadded to the constitutive amino acids, or wherein any of theconstitutive amino acids is substituted by other amino acids includepeptides having the amino acid sequence shown by SEQ ID NO:11, wherein 1to 3, preferably 1 or 2 amino acids are deleted, wherein 1 to 3,preferably 1 or 2 amino acids are added (or inserted), or wherein 1 to3, preferably 1 or 2 amino acids are substituted with other amino acids.

[0078] Furthermore, examples of said peptide wherein any constitutiveamino acids are deleted, peptides wherein other amino acids are added tothe constitutive amino acids, peptides wherein any constitutive aminoacids are substituted by other amino acids include peptides having theamino acid sequence shown by SEQ ID NO:5, NO:6, NO:7, NO:8, NO:9, NO:10,NO:12, NO:13, NO:14 or NO:15, wherein 1 to 3, preferably 1 or 2 aminoacids are deleted, wherein 1 to 3, preferably 1 or 2 amino acids areadded (or inserted), or wherein 1 to 3, preferably 1 or 2 amino acidsare substituted with other amino acids.

[0079] The amino acid in said amino acid sequence can be substitutedwith substantially the same amino acid selected from, for example, otheramino acids of the group that the amino acid belongs to. Non-polar(hydrophobic) amino acid includes alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, methionine, etc. Polar (neutral)amino acid includes glycine, serine, threonin, cycteine, thyrosine,asparagine, glutamine, etc. Positively charged (basic) amino acidincludes arginine, lysine, histidine, etc. Negatively charged (acidic)amino acid includes aspartic acid, glutamic acid, etc.

[0080] Examples of the labeled Neuromedin U, the labeled partial peptidedescribed (1) above and the labeled peptide described (2) above includethose labeled with an isotope, those labeled with fluorescence (e.g.fluorescein), those biotinated, and those labeled with enzyme accoridngto a known method.

[0081] Specifically, Neuromedin U labeled with the radioisotope such as[³H], [¹²⁵I], [¹⁴C], [³⁵S] in a known manner is used. The salt ofNeuromedin U derivatives is the same salt as those salts of TGR-1 orNeuromedin U mentioned above.

[0082] The DNA encoding TGR-1 used in the present invention may be anyDNA encoding the protein having the same or substantially the same aminoacid sequence as that shown by SEQ ID NO:1 or NO:21, and the DNAencoding Neuromedin U used in the present invention may be any DNAencoding the peptide having the same or substantially the same aminoacid sequence as that shown by SEQ ID NO:11. These DNAs may be derivedfrom any of genomic DNA, genomic DNA library, cDNA derived from thecells and tissues described above, cDNA library derived from the cellsand tissues described above, and synthetic DNA. Vectors to be used forthe library may be any of bacteriophage, plasmid, cosmid and phagemid.The DNA may also be directly amplified by reverse transcriptasepolymerase chain reaction (RT-PCR) using the total RNA fraction preparedfrom the cells and tissues described above.

[0083] An example of the DNA encoding TGR-1 having the amino acidsequence shown by SEQ ID NO:1 includes the DNA having the base sequenceshown by SEQ ID NO:2. An example of the DNA encoding TGR-1 having theamino acid sequence shown by SEQ ID NO:21 includes the DNA having thebase sequence shown by SEQ ID NO:22. An example of the DNA encodingTGR-1 having the amino acid sequence shown by SEQ ID NO:17 includes theDNA having the base sequence shown by SEQ ID NO:18.

[0084] Further, an example of a DNA comprising the DNA encoding TGR-1comprising a portion from 4th (Met) to 415th (Thr) position from theN-terminal of the amino acid sequence shown by SEQ ID NO:1 includes theDNA comprising a portion from 10th (A) to 1245th (C) position from the5′-terminal of the nucleic acid sequence shown by SEQ ID NO:2, and anexample of a DNA comprising the DNA encoding TGR-1 comprising a portionfrom 4th (Met) to 415th (Thr) position from the N-terminal of the aminoacid sequence shown by SEQ ID NO:17 includes the DNA comprising aportion from 10th (A) to 1245th (C) position from the 5′-terminal of thenucleic acid sequence shown by SEQ ID NO:18.

[0085] In particular, an example of a DNA comprising the DNA encodingTGR-1 includes a DNA which comprises the nucleic acid sequence-CTGTTTGTC- (a portion from 808th to 816th position of the sequenceshown by SEQ ID NO:2), -TGGAGTGAA- (a portion from 888th to 896thposition of the sequence shown by SEQ ID NO:2), -GTCTTCTTC- (a portionfrom 940th to 948th position of the sequence shown by SEQ ID NO:2), or-TCCATGCAC- (a portion from 1159th to 1167th position of the sequenceshown by SEQ ID NO:2), and preferred is a DNA comprising the nucleicacid sequence shown by SEQ ID NO:2.

[0086] Specifically, the following DNA is used: (1) DNA hybridizingunder high stringent conditions with the DNA encoding the protein or(poly)peptide having the same or substantially the same amino acidsequence as that shown by SEQ ID NO:1, NO:21 or NO:11; (2) DNA whichencodes the protein or (poly)peptide having the same or substantiallythe same amino acid sequence as that shown by SEQ ID NO:1, NO:21 orNO:11, but which does not hybridize with the DNA sequence encoding theprotein or (poly)peptide having the same or substantially the same aminoacid sequence shown by SEQ ID NO:1, NO:21 or NO:11, or the sequencedetermined in (1), due to the degeneracy of genetic code.

[0087] Hybridization can be carried out according to a known method. Thehigh stringent conditions used herein refer to the conditions, forexample, 50% formaldehyde, 4×SSPE (1×SSPE=150 mM NaCl, 10 mMNaH₂PO₄/H₂O, 1 mM EDTA, pH 7.4), 5× Denhardt's solution and 0.1% of SDSat a temperature of 42° C.

[0088] The DNA encoding TGR-1 or Neuromedin U used in the presentinvention can be produced according to a genetic engineering methoddescribed below.

[0089] For cloning the complete DNA encoding TGR-1 or Neuromedin U ofthe present invention, the desired DNA may be amplified by the known PCRmethod using synthetic DNA primers containing a part of the basesequence encoding the polypeptide of the present invention from theabove-mentioned DNA library, or DNAs inserted into an appropriate vectorcan be selected by hybridization with a labeled DNA fragment orsynthetic DNA having a part or whole of the base sequence encoding TGR-1or Neuromedin U. The hybridization can be carried out, for example,according to the method described in Molecular Cloning, 2nd, J. Sambrooket al., Cold Spring Harbor Lab. Press, 1989. The hybridization may alsobe performed using commercially available library in accordance with theprotocol described in the attached instructions.

[0090] Conversion of the base sequence of the DNA can be carried out byknown methods, such as the ODA-LA PCR method, the Gupped duplex methodor the Kunkel method or its modification using a known kit available asMutan™-super Express Km or Mutan™-K (both produced by Takara Shuzo Co.,Ltd.).

[0091] The cloned DNA encoding TGR-1 or Neuromedin U used in the presentinvention can be used depending upon purpose, as it is or if desired,after digestion with a restriction enzyme or after addition of a linkerthereto. The DNA may contain ATG as a translation initiation codon atthe 5′ end thereof and may further contain TAA, TGA or TAG as atranslation termination codon at the 3′ end thereof. These translationinitiation and termination codons can also be added by using anappropriate-synthetic DNA adapter.

[0092] The expression vector for TGR-1 or Neuromedin U used in thepresent invention can be produced, for example, by (a) excising thedesired DNA fragment from the DNA encoding TGR-1 or Neuromedin U of thepresent invention, and then (b) ligating the DNA fragment into anappropriate expression vector downstream of a promoter.

[0093] Examples of the vector include plasmids derived form E. coli(e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillussubtilis (e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g.,pSH19, pSH15), bacteriophages such as λ phage, etc., animal viruses suchas retrovirus, vaccinia virus, baculovirus, etc. The promoter used inthe present invention may be any promoter suitable for a host to be usedfor gene expression.

[0094] When the host is animal cells, SV40 promoter, a retroviruspromoter, a metallothionein promoter, a heat shock promoter, acytomegalovirus promoter, SRα promoter, etc can be used. When the hostis Escherichia bacteria, preferred are trp promoter, T7 promoter, lacpromoter, recA promoter, λP_(L) promoter, lpp promoter, etc. When thehost is Bacillus bacteria, preferred are SPO1 promoter, SPO2 promoterand penP promoter, etc. When the host is yeast, preferred are PHO5promoter, PGK promoter, GAP promoter and ADH1 promoter, GAL promoter,etc. When the host is insect cells, preferred are polyhedrin prompterand P10 promoter, etc.

[0095] In addition, the expression vector may further optionally containan enhancer, a splicing signal, a poly A addition signal, a selectionmarker, SV40 replication origin (hereinafter sometimes abbreviated asSV40ori), etc. Examples of the selection marker include dihydrofolatereductase gene (hereinafter sometimes abbreviated as dhfr) [methotrexate(MTX) resistance], ampicillin resistant gene (hereinafter sometimesabbreviated as Amp^(r)), neomycin resistant gene (hereinafter sometimesabbreviated as Neo^(r), G418 resistance), etc. In particular, when dhfrgene is used as the selection marker in CHO (dhfr⁻) cell, selection canalso be carried out in thymidine free medium.

[0096] If necessary, a signal sequence suitable for a host is added tothe N-terminal of the polypeptide or the partial peptide. Examples ofthe signal sequence that can be used are Pho A signal sequence, OmpAsignal sequence, etc. for an Escherichia bacterium host; α-amylasesignal sequence, subtilisin signal sequence, etc. for a Bacillusbacterium host; MFα signal sequence, invertase signal sequence, etc. fora yeast host; and insulin signal sequence, α-interferon signal sequence,antibody molecule signal sequence, etc. for an animal cell host.

[0097] Using the vector containing the DNA encoding TGR-1 or NeuromedinU of the present invention thus constructed, transformants can beproduced.

[0098] Examples of the host which may be employed, are Escherichiabacteria, Bacillus bacteria, yeast, insect cells, insects and animalcells, etc.

[0099] Examples of the Escherichia bacteria include Escherichia coli K12DH1 (Proc. Natl. Acad. Sci. U.S.A., 60, 160 (1968)), JM103 (NucleicAcids Research, 9, 309 (1981)), JA221 (Journal of Molecular Biology,120, 517 (1978)), HB101 (Journal of Molecular Biology, 41, 459 (1969)),C600 (Genetics, 39, 440 (1954)), etc.

[0100] Examples of the Bacillus bacteria include Bacillus subtilis MI114(Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry, 95, 87 (1984)),etc.

[0101] Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, etc.

[0102] Examples of insect include a larva of Bombyx mori (Maeda, et al.,Nature, 315, 592 (1985)).

[0103] Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cells (Sf cells), MG1 cells derived from mid-intestine ofTrichoplusia ni, High Five™ cells derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cells (BmN cells),etc. Examples of the Sf cell which can be used are Sf9 cells (ATCCCRL1711) and Sf21 cells (both cells are described in Vaughn, J. L. etal., In Vitro, 13, 213-217 (1977).

[0104] Examples of animal cells include monkey cells COS-7, Vero cells,Chinese hamster cells CHO (hereinafter referred to as CHO cells), dhfrgene deficient Chinese hamster cells CHO (hereinafter simply referred toas CHO (dhfr⁻) cell), mouse L cells, mouse 3T3, mouse myeloma cells,human HEK293 cells, human FL cells, 293 cells, C127 cells, BALB3T3cells, Sp-2/O cells, etc.

[0105] Escherichia bacteria can be transformed, for example, by themethod described in Proc. Natl. Acad. Sci. U.S.A., 69, 2110 (1972) orGene, 17, 107 (1982).

[0106] Bacillus bacteria can be transformed, for example, by the methoddescribed in Molecular & General Genetics, 168, 111 (1979).

[0107] Yeast can be transformed, for example, by the method described inProc. Natl. Acad. Sci. U.S.A., 75, 1929 (1978), etc.

[0108] Insect cells or insects can be transformed, for example,according to the method described in Bio/Technology, 6, 47-55(1988),etc.

[0109] Animal cells can be transformed, for example, according to themethod described in Virology, 52, 456 (1973).

[0110] The method of introducing the expression vector into the cellincludes, for example, lipofection (Felgner, P. L. et al. Proc. Natl.Acad. Sci. U.S.A., 84, 7413 (1987)), calcium phosphate method (Graham,F. L. and van der Eb, A. J. Virology, 52, 456-467 (1973)),electroporation (Nuemann, E. et al. Embo J., 1, 841-845 (1982)), etc.

[0111] Thus, the transformant transformed with the expression vectorcontaining the DNA encoding TGR-1 or Neuromedin U can be obtained.

[0112] Furthermore, to express TGR-1 or Neuromedin U used in the presentinvention in a stable manner using animal cells, the animal cell clonecan be selected, into the chromosome of which the introduced expressionvector is incorporated. To be more specific, using the above selectionmarker as an index, a transformant can be selected. From these animalcells obtained by use of the selection marker, it is possible to obtaina stable animal cell strain having a highly expressed TGR-1 orNeuromedin U used in the present invention by repeating the clonalselection. Moreover, when using dhfr gene as a selection marker, thecells are cultured in gradually increased concentrations of MTX, and theresistant cell strain is selected. In this way, it is possible to obtainthe highly expression animal cell strain by amplifying the DNA encodingTGR-1 or Neuromedin U as well as dhfr gene in the cell.

[0113] TGR-1 or Neuromedin U used in the present invention can beproduced by cultivating the above-mentioned transformant under conditioncapable of expressing the DNA encoding TGR-1 or Neuromedin U used in thepresent invention; and producing and accumulating TGR-1 or Neuromedin Uused in the present invention.

[0114] When the host is Escherichia or Bacillus bacteria, thetransformant can be appropriately cultured in a liquid medium, whichcontains materials required for growth of the transformant, such ascarbon sources, nitrogen sources, inorganic materials, and so on.Examples of the carbon sources include glucose, dextrin, soluble starch,sucrose, etc. Examples of the nitrogen sources include inorganic ororganic materials such as ammonium salts, nitrate salts, corn steepliquor, peptone, casein, meat extract, soybean cake, potato extract,etc. Examples of the inorganic materials are calcium chloride, sodiumdihydrogenphosphate, magnesium chloride, etc. In addition, yeast,vitamins, growth promoting factors etc. may be added to the medium.Preferably, pH of the medium is about 5 to 8.

[0115] A preferred example of the medium for culturing Escherichiabacteria is M9 medium supplemented with glucose and Casamino acids(Miller, Journal of Experiments in Molecular Genetics, 431-433, ColdSpring Harbor Laboratory, New York, 1972). If necessary, a chemical suchas 3β-indolylacrylic acid can be added to the medium to work thepromoter efficiently.

[0116] When the host is Escherichia bacteria, the transformant isusually cultivated at about 15° C. to 43° C. for about 3 to 24 hours. Ifnecessary, the culture may be aerated or agitated.

[0117] When the host is Bacillus bacteria, the transformant iscultivated generally at about 30° C. to 40° C. for about 6 to 24 hours.If necessary, the culture can be aerated or agitated.

[0118] When the host is yeast, the transformant is cultivated, forexample, in Burkholder's minimal medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)) or in SD medium supplementedwith 0.5% Casamino acids (Bitter, G. A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)). Preferably, pH of the medium is about 5 to 8.In general, the transformant is cultivated at about 20° C. to 35° C. forabout 24 to 72 hours. If necessary, the culture can be aerated oragitated.

[0119] When the host is insect cells or insects, the transformant iscultivated in, for example, Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) to which an appropriate additive such as 10%inactivated bovine serum is added. Preferably, pH of the medium is about6.2 to 6.4. Normally, the transformant is cultivated at about 27° C. forabout 3 to 5 days and, if necessary, the culture can be aerated oragitated.

[0120] When the host is animal cells, the transformant is cultivated in,for example, MEM medium (Science, 122, 501 (1952)), DMEM medium(Virology, 8, 396 (1959)), RPMI 1640 medium (The Journal of the AmericanMedical Association, 199, 519 (1967)), 199 medium (Proceeding of theSociety for the Biological Medicine, 73, 1 (1950)), which contain about5% to about 20% fetal bovine serum. Preferably, pH of the medium isabout 6 to 8. The transformant is usually cultivated at about 30° C. to40° C. for about 15 to 60 hours and, if necessary, the culture can beaerated or agitated.

[0121] When using CHO (dhfr⁻) cells and dhfr gene as a selection marker,thymidine-free DMEM medium containing dialyzed fetal bovine serum ispreferred.

[0122] TGR-1 or Neuromedin U used in the present invention can beseparated and purified from the culture described above by the followingprocedures.

[0123] When TGR-1 or Neuromedin U used in the present invention isextracted from the cultured transformants or cells, after cultivation,the transformants or cells are collected by a well-known method,suspended in a appropriate buffer, and then disrupted by publicly knownmethods such as ultrasonication, a treatment with lysozyme and/orfreeze-thaw cycling. Then by centrifugation, filtration, etc., the crudeextract of TGR-1 or Neuromedin U used in the present invention can beobtained. The buffer for the extraction may contain a proteindenaturizing agent, such as urea or guanidine hydrochloride, or asurfactant, such as Triton X-100™, etc.

[0124] When TGR-1 or Neuromedin U used in the present invention issecreted to the culture medium, after the cultivation, the transformantsor cells can be separated to collect the supernatant by a well-knownmethod.

[0125] TGR-1 or Neuromedin U present in the supernatant or the extractthus obtained can be purified by an appropriate combination ofwell-known methods for separation and purification. Such publicly knownmethods for separation and purification include a method utilizingdifference in solubility such as salting out, solvent precipitation,etc.; a method utilizing difference mainly in molecular weight such asdialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gelelectrophoresis, etc.; a method utilizing difference in electric chargesuch as ion exchange chromatography, etc.; a method utilizing differencein specific affinity such as affinity chromatography, etc.; a methodutilizing difference in hydrophobicity such as reverse phase highperformance liquid chromatography, etc.; a method utilizing differencein isoelectric point such as isoelectrofocusing, chromatofocusing; andthe like.

[0126] When TGR-1 or Neuromedin U used in the present invention isobtained in a free form, it can be converted into a salt form bywell-known methods or modifications thereof. On the other hand, whenTGR-1 or Neuromedin U is obtained in a salt form, it can be convertedinto the free form or another salt form by well-known methods ormodifications thereof.

[0127] TGR-1 or Neuromedin U used in the present invention produced by arecombinant can be treated, before or after the purification, with anappropriate protein modifying enzyme so that TGR-1 or Neuromedin U canbe appropriately modified or be deprived of a partial (poly)peptide.Examples of the protein-modifying enzyme include trypsin, chymotrypsin,arginyl endopeptidase, protein kinase, glycosidase or the like. It ispossible to use the well-known Edman method using Edman reagent (phenyliso-thiocyanate) to delete the N-terminal amino acid.

[0128] The presence of the thus produced TGR-1 or Neuromedin U used inthe present invention can be determined by an enzyme immunoassay usingan antibody specific thereto, or the like.

[0129] The screening method for a compound or its salt that alters thebinding property between Neuromedin U and TGR-1, characterized by usingNeuromedin U and TGR-1, and the screening kit for a compound or its saltthat alters the binding property between Neuromedin U and TGR-1,characterized by comprising Neuromedin U and TGR-1, are described indetail below.

[0130] Using the binding assay system (ligand/receptor assay system) ofNeuromedin U with TGR-1 or the constructed recombinant TGR-1 expressionsystem, the compound that alters the binding property between NeuromedinU and TGR-1 (e.g., peptides, proteins, non-peptide compounds, syntheticcompounds, fermentation products, etc.) or its salt can be screened.

[0131] These compounds include the compound (TGR-1 agonist) havingTGR-1-mediated cell-stimulating activities (e.g., activities ofenhancing arachidonic acid release, acetylcholine release, intracellularCa²⁺ release, intracellular cAMP production, intracellular cGMPproduction, inositol phosphate production, cell membrane potentialchange, phosphorylation of intracellular proteins, c-fos activation andpH change), and the compound (TGR-1 antagonist) having no suchcell-stimulating activities.

[0132] The wording “alter the binding property between Neuromedin U andTGR-1” means either of properties of inhibiting or enhancing the bindingbetween Neuromedin U and TGR-1 (prolonging the binding time).

[0133] Thus, the present invention provides:

[0134] a method for screening a compound or a salt thereof that altersthe binding property of Neuromedin U with TGR-1, characterized bycomparing (i) a case where Neuromedin U is brought in contact with theabove-mentioned TGR-1 and (ii) a case where Neuromedin U and a testcompound are brought in contact with the TGR-1.

[0135] In the screening method of the present invention, for example, abinding amount of the ligand with the TGR-1 and a level ofcell-stimulating activity (e.g., activities of enhancing arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation and pH change) are measured andcompared (i) where Neuromedin U is brought in contact with theabove-mentioned TGR-1 and (ii) where Neuromedin U and a test compoundare brought in contact with the TGR-1.

[0136] Specifically, the screening method of the present invention is:

[0137] (1) A method for screening a compound or a salt thereof thatalters the binding property between Neuromedin U and TGR-1, whichcomprises measuring and comparing the binding amounts of a labeledNeuromedin U as one of the above-mentioned Neuromedin derivatives(hereinafter simply referred to as “labeled Neuromedin U”) with theabove-mentioned TGR-1, where a labeled Neuromedin U is brought incontact with the TGR-1 and where a labeled Neuromedin U and a testcompound are brought in contact with the TGR-1;

[0138] (2) A method for screening a compound or a salt thereof thatalters the binding property between Neuromedin U and TGR-1, whichcomprises measuring and comparing the binding amounts of a labeledNeuromedin U with a cell containing TGR-1 or a membrane fraction of thecell, where a labeled Neuromedin U is brought in contact with the cellor membrane fraction thereof and where a labeled Neuromedin U and a testcompound are brought in contact with the cell containing TGR-1 ormembrane fraction thereof;

[0139] (3) A method for screening a compound or a salt thereof thatalters the binding property between Neuromedin U and TGR-1, whichcomprises measuring and comparing the binding amounts of a labeledNeuromedin U with TGR-1, where a labeled Neuromedin U is brought incontact with TGR-1 expressed on cell membrane of a cultured transformantcontaining DNA encoding TGR-1 and where a labeled Neuromedin U and atest compound are brought in contact with TGR-1 expressed on cellmembrane of a cultured transformant containing DNA encoding TGR-1;

[0140] (4) A method for screening a compound or a salt thereof thatalters the binding property between Neuromedin U and TGR-1, whichcomprises measuring and comparing TGR-1-mediated cell-stimulatingactivities (e.g., activities of enhancing arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, c-fos activation and pH change), where acompound which activates TGR-1 (e.g. Neuromedin U) is brought in contactwith a cell containing TGR-1 and where a compound which activates TGR-1and a test compound are brought in contact with the cell; and

[0141] (5) A method for screening a compound that or a salt thereofalters the binding property between Neuromedin U and TGR-1, whichcomprises measuring and comparing TGR-1-mediated cell-stimulatingactivities (e.g., activities of enhancing arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation and pH change), where acompound which activates TGR-1 (e.g. Neuromedin U) is brought in contactwith TGR-1 expressed on cell membrane of a cultured transformantcontaining DNA encoding TGR-1 and where a compound which activates TGR-1and a test compound are brought in contact with TGR-1 expressed on thecell membrane of cultured transformant containing DNA encoding TGR-1.

[0142] Neuromedin U used in the present invention is known to have aligand activity to an orphan receptor FM-3 (Tan, C. P. et al., Genomics52, 223-229, 1998)(WO 00/02919). Therefore, it is possible to screen acompound which alters the binding property between Neuromedin U and FM-3(FM-3 antagonist, FM-3 agonist) using FM-3 in place of TGR-1 in theabove mehtods (1)-(5).

[0143] Accordingly, by comparing an activity of TGR-1 antagonist orTGR-1 agonist which is obtained by the screening method of the presentinvention, with an activity of FM-3 antagonist or FM-3 agonist which isobtained by the screening method of the present invention using FM-3 inplace of TGR-1, an antagonist or agonist which acts on FM-3preferentially over TGR-1 or which acts on TGR-1 preferentially overFM-3, can be obtained.

[0144] The term “an antagonist which acts on FM-3 preferentially overTGR-1” refers to a compound or a salt thereof in which FM-3-mediatedcell-stimulating activities (e.g. activities of enhancing arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation, and pH change) are at least 2times, preferably at least 10 times weaker than TGR-1-mediatedactivities (receptor (TGR-1, FM-3)).

[0145] The term “an agonist which acts on FM-3 preferentially overTGR-1” refers to a compound or a salt thereof in which FM-3-mediatedcell-stimulating activities (e.g. activities of enhancing arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation, and pH change) are at least 2times, preferably at least 10 times stronger than TGR-1-mediatedactivities (receptor (TGR-1, FM-3)).

[0146] The term “an antagonist which acts on TGR-1 preferentially overFM-3” refers to a compound or a salt thereof in which TGR-1-mediatedcell-stimulating activities (e.g. activities of enhancing arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation, and pH change) are at least 2times, preferably at least 10 times weaker than FM-3-mediated activities(receptor (TGR-1, FM-3)).

[0147] The term “an agonist which acts on TGR-1 preferentially overFM-3” refers to a compound or a salt thereof in which TGR-1-mediatedcell-stimulating activities (e.g. activities of enhancing arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, cell membrane potential change, phosphorylation ofintracellular proteins, c-fos activation, and pH change) are at least 2times, preferably at least 10 times stronger than FM-3-mediatedactivities (receptor (TGR-1, FM-3)).

[0148] The concrete description of the screening methods of the presentinvention is as follows.

[0149] For the TGR-1 used in the screening method of the presentinvention, any substance may be used so long as it contains theabove-mentioned TGR-1. The cell membrane fraction from organs of human,warm-blooded animal, amphibian or fish is preferred. Because organs, inparticular human organs, are very difficult to obtain, it is preferableto use TGR-1 produced by a recombinant in a large scale. To produceTGR-1, the above-mentioned methods may be applied.

[0150] In the screening methods, the cell containing TGR-1 or the cellmembrane fraction can be prepared according to the preparation methoddescribed below.

[0151] When the cells containing TGR-1 are used, the cells may be fixedusing glutaraldehyde, formalin, etc. The fixation can be made by awell-known method.

[0152] The cells containing TGR-1 include host cells that have expressedTGR-1. Such host cells include Escherichia coli, Bacillus subtilis,yeast, insect cells, animal cells, and the like, as described above.

[0153] The cell membrane fraction refers to a fraction abundant in cellmembrane, obtained by cell disruption and subsequent fractionation by awell-known method. The cell disruption methods include cell squashingusing a Potter-Elvehjem homogenizer, disruption using a Waring blenderor Polytron (produced by Kinematica Inc.), disruption byultrasonication, and disruption by cell spraying through thin nozzlesunder an increased pressure using a French press or the like. Cellmembrane fractionation is carried out mainly by fractionation using acentrifugal force, such as centrifugal fractionation or density gradientcentrifugation. For example, after the disrupted cell solution iscentrifuged at a low speed (500 rpm to 3,000 rpm) for a short period(normally about 1 to 10 minutes), the resulting supernatant iscentrifuged at a higher speed (15,000 rpm to 30,000 rpm) normally for 30minutes to 2 hours. The precipitate thus obtained is used as themembrane fraction. The membrane fraction is rich in expressed TGR-1, andmembrane components, such as cell-derived phospholipids and membraneproteins.

[0154] The amount of TGR-1 in the cell containing TGR-1 and in themembrane fraction is preferably 10³ to 10⁸ molecules per cell, morepreferably 10⁵ to 10⁷ molecules per cell. As the amount of expressedTGR-1 increases, the ligand binding activity per unit of membranefraction (specific activity) increases so that not only the highlysensitive screening system can be constructed but also large quantitiesof samples can be assayed with the same lot.

[0155] To perform the methods (1) through (3) described above forscreening a compound that alters the binding property between NeuromedinU and TGR-1, an appropriate TGR-1 fraction, a labeled ligand or acompound having a ligand activity (e.g. Neuromedin U and a salt thereof)are used. TGR-1 fraction is preferably a fraction of a naturallyoccurring receptor protein or a recombinant receptor protein having anactivity equivalent to that of the natural protein. Herein, theequivalent activity is intended to mean a ligand binding activity. Forthe labeled ligand and the compound having a ligand activity (e.g.Neuromedin U and a derivative thereof), for example, a ligand labeledwith [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. (a labeled Neuromedin U as aNeuromedin U derivative) are used.

[0156] More specifically, to perform the screening for a compound thatalters the binding property between Neuromedin U and TGR-1, first, areceptor preparation is prepared by suspending cells containing TGR-1 orthe membrane fraction thereof in a buffer appropriate for the screeningmethod. Any buffer can be used so long as it does not inhibit theligand-receptor binding, such buffers including a phosphate buffer or aTris-HCl buffer having pH of 4 to 10 (preferably pH of 6 to 8). For thepurpose of minimizing non-specific binding, a surfactant such as CHAPS,Tween-80™ (Kao-Atlas Inc.), digitonin or deoxycholate, may optionally beadded to the buffer. Further for the purpose of suppressing thedegradation of TGR-1 and Neuromedin U by proteases, a protease inhibitorsuch as PMSF, leupeptin, E-64 (Peptide Institute, Inc.) and pepstatinmay also be added. A given amount (5,000 to 500,000 cpm) of labeledNeuromedin U (a Neuromedin U derivative) is added to 0.01 ml to 10 ml ofthe receptor solution. Also, 10⁻⁴ to 10⁻¹ iM of the test compound areadded to the mixture. To determine the amount of non-specific binding(NSB), a reaction tube containing an excessive amount of unlabeledNeuromedin U is also prepared. The reaction is carried out atapproximately 0 to 50° C., preferably 4 to 37° C. for 20 minutes to 24hours, preferably 30 minutes to 3 hours. After completion of thereaction, the reaction mixture is filtrated through glass fiber filterpaper, etc. and washed with an appropriate volume of the buffer. Theresidual radioactivity on the glass fiber filter paper is then measuredwith a liquid scintillation counter or γ-counter. Where regarding thecount obtained by subtracting the amount of non-specific binding (NSB)from the count obtained in the absence of any competitive substance (BO)as 100%, the test compound which makes the specific binding amount(B-NSB), for example 80% or less, can be selected as the compoundcapable of altering the binding property between TGR-1 and Neuromedin U.

[0157] For measuring the binding between TGR-1 and Neuromedin U, BIAcore(Amasham pharmacia Biotech) may be used. In this method, Neuromedin U isfixed to a sensor chip according to amino coupling method described inthe protocol that is attached to the device. A buffer (such as phosphatebuffer and Tris buffer) solution containing TGR-1 purified from thecells having TGR-1 or a transformant having the DNA encoding TGR-1, or amembrane fraction having TGR-1, or a buffer solution containing apurified TGR-1 or a membrane fraction having TGR-1 and a test compoundis flowed on the top of the sensor chip at 2-20 μl/min. By investigatingwhether the co-existing test compound can alter the surface plasmoresonance change which is induced by binding TGR-1 to Neuromedin U onthe sensor chip, the compound that alters the binding property betweenTGR-1 and Neuromedin U can be screened. This method can also be carriedout by fixing TGR-1 to the sensor chip and flowing the buffer solution(such as phosphate buffer or Tris buffer) containing Neuromedin U and atest compound on the top of the sensor chip. These test compounds are asdescribed above.

[0158] To perform the above screening methods (4) and (5) for thecompound that alters the binding property between Neuromedin U andTGR-1, TGR-1-mediated cell-stimulating activity (e.g., activities ofpromoting or inhibiting arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, cell membrane potentialchange, phosphorylation of intracellular proteins, c-fos activation, pHchange, etc.) can be measured using known methods or commerciallyavailable measuring kits. Specifically, the cells containing TGR-1 arefirst cultured on a multi-well plate. For the screening, the medium isreplaced with fresh medium or with an appropriate non-cytotoxic buffer,followed by incubation for a given period of time in the presence of atest compound. Subsequently, the resulting product is quantified byappropriate procedures in the cell extract or the supernatant. When itis difficult to detect the production of the index substance (e.g.,arachidonic acid) for the cell-stimulating activity, due to a degradingenzyme present in the cells, an inhibitor against such a degradingenzyme may be added before the assay. For detecting an inhibitoryactivity, such as the inhibition of cAMP production, the basicproduction in the cells can be increased by forskolin or the like andthen the inhibitory effect on the increased basic production can bedetected.

[0159] The screening by assay of the cell-stimulating activity requiresan appropriate cell expressing TGR-1. For the cell expressing TGR-1, therecombinant cell expressing the TGR-1 described above and the like aredesirable. The transformed cells capable of expressing TGR-1 can beeither a stable expression strain or a transient expression strain. Thesame kinds of animal cells described above are used.

[0160] For the test compound, for example, peptides, proteins,non-peptide compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, and animal tissue extracts are used.

[0161] To describe the above-mentioned ligand/receptor assay morespecifically, the following assay systems and the like are used.

[0162] [1] When a receptor-expressing cell is stimulated by a receptoragonist, an intracellular G-protein becomes active and, as a result, GTPbonds with it. The same phenomena can be observed with a cell membraneof receptor expression cell. Generally, GTP is converted to GDP byhydrolysis. When GTPγS is added to the reaction solution, GTPγS bondswith G-protein as GTP does, and it does not suffer from hydrolysis withkeeping the binding to the cell membrane containing the G-protein. Usingthe labeled GTPγs, it is possible to measure the receptor expressioncell stimulating activity of the receptor agonist by measuring theradioactivity remaining in the cell membrane. Applying this reaction, astimulating activity of Neuromedin U with respect to TGR-1-expressingcells can be measured. This method does not use the cells containingTGR-1 as described above (4)-(5). This method is an assay using the cellmembrane containing TGR-1 as described in (1)-(3), and is an assay tomeasure a cell stimulating activity as described in (4)-(5). In thisassay, a substance which shows an activity to promote the binding ofGTPγS to TGR-1-containing cell membrane fraction is an agonist. Byadding Neuromedin U or Neuromedin U and a test compound and observingthe change in GTPγS binding acceleration activity to a TGR-1-containingcell membrane fraction as compared with a single administration ofNeuromedin U, the compound that alters the binding property betweenNeuromedin U and TGR-1 can be screened. The compound which indicates theactivity that inhibits the GTPγS binding acceleration activity to aTGR-1-containing cell membrane fraction by Neuromedin U can be selectedas a candidate substance which is capable of altering the bindingproperty between TGR-1 and Neuromedin U. On the other hand, an agonistcan be screened by adding a test compound alone and observing the GTPγSbinding acceleration activity to a TGR-1-containing cell membranefraction as well.

[0163] Concretely, an example of the screening methods is described asfollows. A cell membrane fraction containing TGR-1 prepared by themethod described above is diluted with a membrane dilution buffersolution (e.g. 50 mM Tris, 5 mM MgCl₂, 150 mM NaCl, 1μ GDP, 0.1% BSA pH7.4). The dilution scale may vary according to the amount of receptorexpression. 0.2 ml of the solution is transferred to Falconb 2053.Neuromedin U or Neuromedin U and a test compound are added thereto, andthen [³⁵S]GTPγS is added to make the final concentration of 200 pM.After the mixture is kept at 25° C. for an hour, an ice-cold buffersolution for washing (50 mM Tris, 5 mM MgCl₂, 150 mM NaCl, 0.1% BSA,0.05% CHAPS pH 7.4 1.5 ml) is added. Then, the solution is filtered witha glass fiber filtering paper GF/F. After drying the filtering paper at65° C. for 30 min., the radioactivity of [³⁵S]GTPγS bound with themembrane fraction left on the filtering paper is measured on a liquidscintillation counter. The radioactivity in the experiment with a singleadministration of Neuromedin U is set as 100%, the radioactivity in theexperiment without adding Neuromedin U is set as 0%, and an influence ofa test compound to the GTPγS binding acceleration activity by NeuromedinU is calculated. A test compound which makes GTPγS binding accelerationactivity, for example 80% or less, can be selected as a candidatesubstance which is capable of altering the binding property betweenTGR-1 and Neuromedin U.

[0164] [2] When the amount of intracellular cAMP is reduced by theNeuromedin U stimulation in a TGR-1-expressing cell, using thisreaction, the cell stimulating activities of Neuromedin to aTGR-1-expressing cell can be measured. Using the anti-cAMP antibodyobtained by immunized mice, rats, rabbits, goats and cows and¹²⁵I-labeled cAMP (both are commercially available), the amount of cAMPproduction in various animal cells expressing TGR-1 can be measured byRIA or other EIA system such as the combination of anti-cAMP antibodyand the labeled cAMP. It is also possible to conduct a quantification bythe SPA method using beads containing the scintillant to which ananti-cAMP is fixed using Protein A or an antibody to IgG of an animalused for production of the anti-cAMP antibody, and ¹²⁵I-labeled cAMP(using the kit produced by Amasham pharmacia Biotech).

[0165] In this assay system, it is possible to conduct a screening ofthe compound that alters the binding property of Neuromedin U and TGR-1by increasing the amount of intracellular cAMP by ligand such asCalcitonin and Forskolin which increase the amount of intracellularcAMP; adding Neuromedin U or Neuromedin U and the test compound; andobserving the change in the amount of intracellular cAMP as compared tothe case with a single administration of Neuromedin U. Then, a compoundthat shows an inhibitory activity on the cAMP production inhibitioninduced by Neuromedin U in the TGR-1-expressing cells can be selected asa candidate substance that is capable of altering the binding propertybetween TGR-1 and Neuromedin U. On the other hand, the compound thatindicates an agonist activity can be screened by adding the testcompound alone and measuring the cAMP production inhibition activity.

[0166] More specifically, the screening methods are described asfollows. TGR-1-expressing CHO cells are plated at 5×10⁴ cell/well on a24-well plate, and cultivated for about 48 hours. The cells are washedwith Hanks' buffer containing 0.2 mM 3-isobutylmethylxanthine, 0.05% BSAand 20 mM HEPES (pH 7.4)(hereinafter referred to as reaction buffer).Then, 0.5 ml of the reaction buffer is added to the cells, and the cellsare kept in an incubator for 30 minutes. Then, the reaction buffer isremoved and 0.25 ml of fresh reaction buffer is added to the cells.Then, the reaction buffer (0.25 ml) containing 2 μM Forskolin inaddition to 1 nM of Neuromedin U or Neuromedin U and a test compound isadded to the cells. The reaction is made at 37° C. for 24 minutes. 100μl of 20% Perchloric acid is added to stop the reaction. Then, byplacing it on ice, the intracellular cAMP is extracted. The amount ofcAMP in the extraction is measured by using cAMP EIA kit (Amashampharmacia biotech). The amount of cAMP produced by the Forskolinstimulation is set as 100%, the amount of cAMP inhibited by the additionof 1 nM of Neuromedin is set as 0% and an influence of the test compoundon the cAMP production inhibition activity by Neuromedin U iscalculated. A test compound which makes the cAMP production activity,for example, 80% or less by inhibiting the Neuromedin U activity can beselected as a candidate substance that is capable of altering thebinding property between TGR-1 and Neuromedin U.

[0167] To measure the cAMP production acceleration activity, the cAMPproduced by adding a test compound to the TGR-1-expressing CHO cellswithout added Forskolin is measured according to the above-mentionedmethod. In this case, a test compound which makes the cAMP productionactivity, for example, 10% or more, can be selected as a candidatesubstance that is capable of altering the binding property between TGR-1and Neuromedin U.

[0168] [3] The DNA containing CRE (cAMP response element) is insertedinto the multi-cloning site upstream of luciferase gene of Picagenebasic vector or Picagene enhancer vector (Toyo Ink). It is named asCRE-reporter gene vector. In the cell transfected with the CRE-reportergene vector, a stimulation which causes the increase in cAMP, induces anexpression of luciferase gene through CRE and a production of luciferaseprotein. By measuring the luciferase activity, it is possible to detectthe change in the amount of cAMP in the cells into which theCRE-reporter gene vector is introduced. Thus, the compound that altersthe binding property of Neuromedin U and TGR-1 can be screened using theTGR-1-expressing cells to which the CRE-reporter gene vector istransfected. The details of the screening method are as follows.

[0169] CRE-reporter gene introduced TGR-1-expressing cells is placed ina 24-well plate at a concentration of 5×10³ cell/well, and cultivatedfor about 48 hours. The cells are washed with Hanks' buffer (pH 7.4)containing 0.2 mM 3-isobutyl-methyl xanthine, 0.05% BSA and 20 mM HEPES(hereinafter, Hanks' buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES, is referred to as reaction buffer)0.5 ml of the reaction buffer is added to the cells. Then, the cells arekept warm in a cultivator for 30 minutes. Then, the reaction buffer isremoved from the system. 0.25 ml of fresh reaction buffer is added tothe cells. Then, the reaction buffer 0.25 ml containing 2 μM Forskolinin addition to 1 nM of Neuromedin U or Neuromedin U and a test compoundis added to the cells. The reaction is made at 37° C. for 24 minutes.The cells are dissolved in a decomposition solution for Picagene (ToyoInk). To the decomposition solution, a luminescent substance (Toyo Ink)is added. The luminescence by luciferases is measured with aluminometor, a liquid scintillation counter, a top counter or the like.An influence of the compound that alters the binding property ofNeuromedin U and TGR-1 can be measured by comparing the luminescence byluciferases with the case where Neuromedin U is singly administrated. Inthis process, by administrating Neuromedin U, the increase ofluminescence by the Folskolin stimulation is inhibited. The compoundthat recovers the influence of Neuromedin U, may be selected as acandidate substance that alters the binding property between NeuromedinU and TGR-1. On the other hand, an agonist can be screened by adding thetest compound singly and observing the inhibition of the increase inluminescence caused by the Folskolin stimulation, as Neuromedin Uinhibits the increase.

[0170] Alkaline phosphatase, chloramphenicol, acetyltransferase orβ-galactosidase can be used as a reporter gene, besides luciferase. Theactivity of the product of reporter gene can be measured easily usingcommercially available measuring kit. The activity of alkalinephosphatase can be measured by Lumi-Phos 530 (Wako); the activity ofChloramphenicol and acetyltransferase can be measured by FAST CATchrolamphenicol Acetyltransferase Assay Kit (Wako); and the activity ofβ-galactosidase can be measured by Aurora Gal-XE (Wako).

[0171] [4] When TGR-1-expressing cells release the metabolic substanceof arachidonic acid to the outside by the Neuromedin stimulation, ifarachidonic acid having radioactivity is taken into the cell beforehand,it is possible to measure a cell stimulating activity by measuring theradioactivity released out of the cells. In this process, by addingNeuromedin U or Neuromedin U and a test compound and examining aninfluence of Neuromedin U on the arachidonic acid metabolite releaseactivity, the compound that alters the binding property betweenNeuromedin U and TGR-1 can be screened. The compound that inhibits thearachidonic acid metabolite release activity of Neuromedin U can beselected as a candidate substance that alters the binding property ofNeuromedin U and TGR-1. Moreover, the compound that indicates an agonistactivity can be screened by adding the test compound singly and checkingthe arachidonic acid metabolite release activity in TGR-1-expressingcells.

[0172] The details of the screening method of the compound that has theinfluence on the binding between Neuromedin U and TGR-1 are as follows.

[0173] TGR-1-expressing CHO cells are placed at 5×10⁴ cell/well on a24-well plate, and cultivated for about 24 hours. After cultivation,0.25 μCi/well of [³H] arachidonic acid is added. 16 hours after adding[³H] arachidonic acid, the cells are washed with Hanks' buffer (pH 7.4)containing 0.05% BSA and 20 mM HEPES. Then, 500 μl of the Hanks' buffer(pH 7.4) containing 0.05% BSA and 20 mM HEPES in the presence of thefinal concentration of 10 nM Neuromedin U or 10 nM Neuromedin U and thetest compound is added to each well (hereinafter, Hanks' buffer (pH 7.4)containing 0.05% BSA and 20 mM HEPES is referred to as reaction buffer).After incubating at 37° C. for 60 minutes, 400 μl of the reactionsolution is added to a scintillator. Then, the amount of released [H³]arachidonic acid metabolite is measured by a scintilation counter. Theamount of [H³] arachidonic acid metabolite in the medium without addedNeuromedin U, is set as 0%, the amount of [H³] arachidonic acidmetabolite in the medium with added 10 nM Neuromedin, is set as 100%,and an influence of the test compound on the binding of Neuromedin U andTGR-1 is calculated. A test compound which makes the arachidonic acidmetabolite production activity, for example, 50% or less, can beselected as a candidate substance that is capable of altering thebinding property between TGR-1 and Neuromedin U.

[0174] [5] When Neuromedin U stimulates the increase in intracellularCa²⁺ concentration in TGR-1-expressing cells, using this fact, aninfluence of test compound on the binding between Neuromedin U and TGR-1can be examined.

[0175] TGR-1-expressing cells are placed on a sterilized cover glass fora microscope. After 2 days, the medium is replaced with HBSS in which 4mM of Fura-2 AM (Dojin Kagaku) is suspended, and left for 2 and halfhours at room temperature. After washing with HBSS, the cover glass isset to a cuvet. The increase in the ratio of intensity of fluorescenceat 505 nm where the excited wave length is 340 nm and 380 nm, ismeasured by a spectrophotofluorometer when Neuromedin U or Neuromedin Uand a test compound are added. By measuring the change in the intensityof fluorescence caused by adding the test compound compared with that bythe single administration of Neuromedin U, the compound which has theinfluence on the binding between Neuromedin U and TGR-1 can be screened.Furthermore, FLIPR (Produced by Molecular device) can be also used asfollows. Fluo-3 AM (Produced by Dojin Kagaku) is added to the cellsuspension to let the cells take up Fluo-3AM. The cells are washed bycentrifuging several times, and placed on a 96-well plate. The cells areset to a FLIPR device, and Neuromedin U or Neuromedin U and a testcompound are added in the same way as Fura-2AM. By measuring the changein the intensity of fluorescence caused by added the test compound ascompared with that by the single administration of Neuromedin U, thecompound which has an influence on the binding between Neuromedin U andTGR-1 can be screened. Above these, the compound that inhibits theincrease in the intensity of fluorescence by Neuromedin U can beselected as a candidate substance that is capable of altering thebinding property between TGR-1 and Neuromedin U. On the other hand, byobserving the increase in the intensity of fluorescence by singleadministration of the test compound, an agonist can be screened.

[0176] To screen the compound that has an influence on the bindingbetween Neuromedin U and TGR-1, first, TGR-1-expressing cells areallowed to co-express a gene of a protein such as Aequorin whichradiates light when the intracellular Ca ion increases. The increase inthe intracellular Ca ion causes Aequorin to become Ca binding type andradiates light. Using this fact, Neuromedin U or Neuromedin U and a testcompound are added and the change in the intensity of luminescence whenthe test compound is added as compared with that by a singleadministration of Neuromedin U is observed for the screening. The methodis almost the same as the above-mentioned method except that this methoddoes not require cells to take up a fluorescence substance.

[0177] [6] By adding an agonist to receptor-expressing cells, theconcentration of inositol triphosphate rises. By observing the reactionin TGR-1-expressing cells caused by Neuromedin U, the compound that hasan influence on the binding between Neuromedin U and TGR-1 can bescreened. Cells are placed in a 24-well plate, and incubated for oneday, and incubated for one more day in a medium to whichmyo-[2-³H]inositol (2.5 μCi/well) is added. After washing well,Neuromedin U or Neuromedin U and a test compound are added thereto, andthen 10% Perchloric acid is added to stop the reaction. The reactionsolution is neutralized with 1.5M KOH and 60 mM HEPES solution, andpassed through a column filled with AG1x8 resin (Bio Rad). After washingwith 5 mM Na₂BO₃ and 60 mM HCOONH₄, the radioactivity, which is elutedby 1M HCOONH₄ and 0.1M HCOOH, is measured by a liquid scintillationcounter. The radioactivity in the medium when Neuromedin U is not added,is set as 0%, the radioactivity in the medium when Neuromedin U isadded, is set as 100%, and an influence on the binding betweenNeuromedin U and TGR-1 can be calculated. A test compound which makesthe inositol triphosphate production activity, for example, 50% or lesscan be selected as a candidate substance that is capable of altering thebinding property between TGR-1 and Neuromedin U. On the other hand, byobserving the increase in the inositol triphosphate production activityby single administration of the test compound, an agonist can bescreened.

[0178] [7] The DNA containing TRE (TPA response element) is insertedinto the multi-cloning site upstream of luciferase gene of Picagenebasic vector or Picagene enhancer vector (Toyo Ink). It is named asTRE-reporter gene vector. In the cell transfected with the TRE-reportergene vector, a stimulation which causes the increase in intracellularCa²⁺ induces an expression of luciferase gene through TRE and aproduction of luciferase protein. By measuring the luciferase activity,it is possible to detect the change in the amount of intracellularcalcium in the cells into which the TRE-reporter gene vector isintroduced. The details of the screening method of the compound thatalters the binding between Neuromedin U and TGR-1 using theTGR-1-expressing cells to which TRE-reporter gene vector is transfectedare as follows.

[0179] The TRE-reporter gene introduced TGR-1-expressing cells areplaced in a 24-well plate at 5×10³ cell/well, and cultivated for about48 hours. The cells are washed with Hanks' buffer (pH 7.4) containing0.05% BSA and 20 mM HEPES. 10 nM Neuromedin or 10 nM Neuromedin U and atest compound are added thereto. Then, the reaction is made at 37° C.for 60 minutes. The cells are dissolved in a decomposition solution forPicagene (Toyo Ink). To the decomposition solution, a luminescencesubstance (Toyo Ink) is added. The luminescence by luciferases can bemeasured with a luminometor, a liquid scintillation counter, a topcounter or the like. An influence of the compound that alters thebinding property of Neuromedin U and TGR-1 can be measured by comparingthe luminescence by luciferases with that when Neuromedin U is singlyadministrated. In this process, by administrating Neuromedin U, theamount of luminescence increases through the increase in intracellularCa²⁺. The compound that inhibits the increase may be selected as acandidate substance that alters the binding property between NeuromedinU and TGR-1. On the other hand, an agonist can be screened by adding thetest compound singly and observing the increase in luminescence like theincrease by Neuromedin U.

[0180] Alkaline phosphatase, chloramphenicol, acyltransferase orβ-galactosidase can be used as a reporter gene, besides luciferase. Theactivity of the product of reporter gene can be measured easily usingcommercially available measuring kit. The activity of alkalinephosphatase can be measured by Lumi-Phos 530 (Wako); the activity ofChloramphenicol and acetyltransferase can be measured by FAST CATchrolamphenicol Acetyltransferase Assay Kit (Wako); and the activity ofβ-galactosidase can be measured by Aurora Gal-XE (Wako).

[0181] [8] If the growth of TGR-1-expressing cells in response toNeuromedin U through the activation of MAP kinase can be observed, thisgrowth can be quantified by measuring the activation of MAP kinase,thymidine incorporation, number of cells (e.g. MTT). Using thesemeasurements, the compound that alters the binding between TGR-1 andNeuromedin U can be screened.

[0182] After adding Neuromedin U or Neuromedin U and a test compound tothe cells, and then obtaining MAP kinase fraction from a decomposed cellsolution by immunoprecipitation with an anti-MAP kinase antibody, MAPkinase activity can be measured easily by using, for example, MAP KinaseAssay Kit (Wako) and γ-[³²P]-ATP. For thymidine incorporation activity,Neuromedin U or Neuromedin U and the test compound are added to theinoculated TGR-1-expressing cells. Then, [methyl-³H]-thymidine is addedthereto. A radioactivity of a labeled thymidine that is taken up intothe cells can be measured by dissolving the cells and counting theradioactivity with a liquid scintillation counter.

[0183] To measure the growth of TGR-1-expressing cells, the cells areinoculated at first, and then Neuromedin U or Neuromedin U and the testcompound are added to the cells. Then, MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) isadded thereto. After dissolving the cells in iso-propanol whichacidified by hydrochloric acid, MTT fromazan which is formed from MTT inthe cells was measured by absorption at 570 nm.

[0184] The details of screening method using the labeled thymidineincorporation activity for the compound that alters the binding betweenTGR-1 and Neuromedin U are as follows.

[0185] TGR-1-expressing cells are placed at 5×10³ cell/well on a 24-wellplate and cultivated for a day. Then, the cells are cultivated in themedium without serum to make the cells to become starved condition.Neuromedin U or Neuromedin U and a test compound are added to the cellsand the cells are cultivated for 24 hours. [methyl-³H]-thymidine at0.015 MBq/well is added thereto and the cells are cultivated for 6hours. The cells are washed with PBS(−), methanol is added thereto andkept still for 10 minutes. Then, 5% trichloro acetate was added and keptstill for 15 minutes. The fixed cells are washed with distilled water 4times. The cells are dissolved in 0.3 N sodium hydroxide. Aradioactivity in the decomposed cell solution is measured with a liquidscintillation counter. An influence of the compound that alters thebinding between Neuromedin U and TGR-1 can be measured by comparing theincrease in the radioactivity in thymidine incorporation with the casewith the single administration of Neuromedin U. The compound thatinhibits the increase in the radioactivity by Neuromedin Uadministration can be selected as a candidate substance that is capableof altering the binding property between Neuromedin U and TGR-1. On theother hand, by administrating the test compound singly and observing theincrease in the radioactivity like that with Neuromedin U, an agonistcan be screened.

[0186] [9] On adding Neuromedin U to TGR-1-expressing cells, K-channelbecomes activated, and K ions in the cells flow out of the cells. Atthis time, Rb ions which belong to the related element, flow out of thecells through K channel as well as K ions. A labeled Rb ([⁸⁶RB]) isadded to the cells to make the cells incorporate it. Then, by measuringthe efflux of [⁸⁶RB], the activity of Neuromedin U can be measured. Thedetails of screening method for the compound that alters the bindingbetween Neuromedin U and TGR-1 by using the efflux activity of [⁸⁶RB]are as follows.

[0187] Two days after placing in a 24-well plate, TGR-1-expressing cellsare kept warm for 2 hours in the medium containing ⁸⁶RBCl (1 mCi/ml).The cells were washed well to remove ⁸⁶RBCl completely from theextracellular solution. Neuromedin U or Neuromedin U and a test compoundare added to the cells, and the extracellular solution is collectedafter 30 minutes. A radioactivity therein is measured by a γ-counter. Aninfluence of the compound that alters the binding between Neuromedin Uand TGR-1 can be measured by comparing the increase in the radioactivityby efflux of [⁸⁶RB] with the case of a single administration ofNeuromedin U. The compound that inhibits the increase in theradioactivity by administrating Neuromedin U, can be selected as acandidate substance that is capable of altering the binding propertybetween Neuromedin U and TGR-1. On the other hand, by administrating thetest compound singly and by observing the increase in the radioactivitylike that by Neuromedin U, an agonist can be screened.

[0188] [10] TGR-1-expressing cells changes extracellular pH(acidification rate) in response to Neuromedin U. By measuring suchchange with a site sensor device (Molecular Device), the activity ofNeuromedin U can be measured. The details of screening method for thecompound that alters the binding between Neuromedin U and TGR-1 bymeasuring the extracellular pH change with the site sensor device are asfollows.

[0189] TGR-1-expressing cells are cultivated in a capsule of the sitesensor over night. The cells are set to the chamber of the device andthey are refluxed with RMPI1640 medium supplemented with 0.1% BSA(Molecular Divice) for 2 hours until the extracellular pH become stable.After the pH becomes stable, measured is the pH change of the mediumcaused by refluxing the medium containing Neuromedin U or Neuromedin Uand a test compound on the cells. An influence of the compound thatalter the binding of Neuromedin U and TGR-1 can be measured by comparingthe change of extracellular pH in TGR-1-expressing cells with that bythe single administration of Neuromedin U. The compound that inhibitsthe change of extracellular pH by administrating Neuromedin U can beselected as a candidate substance that is capable of altering thebinding property between Neuromedin U and TGR-1. On the other hand, byadministrating the test compound singly and observing the extracellularpH change like that by Neuromedin U, an agonist can be screened.

[0190] [11] A sex pheromone receptor STe2 of haploid α-mating Type (MATα) of yeast (Saccharomyces cerevisiae) is coupled with G-protein Gpa1.In response to sex pheromone α-mating factor, the receptor activates MAPkinase, and sequentially Far1 (cell-cycle arrest) and transcriptionactivation factor Ste12. Ste12 induces the expression of variousproteins related to the mating, including FUSI. On the other hand, theregulatory factor Sst2 works in an inhibitory manner in the aboveprocess. In this system, yeast into which the receptor gene isintroduced is prepared. The intracellular signal transduction system inthe yeast is activated by a receptor agonist stimulation, and anexperiment for the measurement system of the reaction between thereceptor agonist and the receptor is conducted by using the growth, etc.resulted from the activation of the intracellular signal transduction asan index (Pausch, M. H., Treinds in Biotechnology, vol.15, pp. 487-494(1997)). Using such system of the receptor gene introduced yeast, thecompound that alters the binding between Neuromedin U and TGR-1 can bescreened.

[0191] The genes encoding Ste2 and GpaI of MATα yeast are removed andthe genes encoding TGR-1 and Gpa1-Gai2 fused protein are introducedinstead. The gene encoding Far is removed to prevent cell cycle arrestand the gene encoding Sst is removed to increase the sensitivity ofresponse to Neuromedin U. Moreover, the FUS1-H1S3 gene in which FUS1 isconnected with a histidine biosynthesis gene HIS3 is introduced. Theabove-mentioned genetic recombinant method can be easily carried outaccording to, for example, the method reported by Price (Price, L. A. etal., Molecular and Cellular Biology, vol. 15, pp.6188-6195 (1995)),using TGR-1 gene in place of a somatostatin receptor type 2 (SSTR2). Thetransformant of yeast constructed according to the above-mentionedmethod reacts to Neuromedin U that is a ligand of TGR-1 with a highsensitivity, causing the activation of MAP kinase and production of ahistidine biosynthetic enzyme so that it can grow in a histidinedeficient medium. Using this system, the response of TGR-1 expressingyeast to Neuromedin U can be observed by using the growth of yeast inthe histidine deficient medium as an index. The screening method for thecompound that alters the binding between Neuromedin U and TGR-1 is asfollows.

[0192] The above-prepared transformant of yeast is cultured in acomplete synthetic medium liquid overnight, added at a concentration of2×10⁴ cell/ml to a melted agar from which histidine is removed, andplated on square Petri dish (9×9 cm). After the agar becomes hard, asterilized filter paper absorbing Neuromedin U or Neuromedin U and atest compound, is placed on the surface of agar and the transformant iscultured for 3 days at 30° C. An influence of the compound that altersthe binding of Neuromedin U and TGR-1 can be measured by comparing thegrowth of yeast around the filter paper with the case of singladministration of Neuromedin U. The compound that inhibits the growth ofyeast by Neuromedin U administration can be selected as a candidatesubstance that is capable of altering the binding property of NeuromedinU and TGR-1. On the other hand, an agonist can be screened byadministrating only the test compound and observing the growth of yeastlike the growth observed in Neuromedin U administration. Furthermore,the transformant of yeast is cultured on the agar containing NeuromedinU, and by observing an influence on the growth of yeast over the surfacein Petri dish around the filter paper absorbing a test compound, aninfluence of the compound that alters the binding of Neuromedin U andTGR-1 can be measured.

[0193] [12] An oocyte of Xenopus Laevis is injected with RNA of TGR-1gene and stimulated by Neuromedin U. As a result, intracellular Ca²⁺concentration increases and calcium-activated chloride current occurs.This change can be detected as a change of membrane potential (similarto the case where K ion concentration gradient is changed). By observingthe reaction caused in the TGR-1-introduced Xenopus Laevis oocytes byNeuromedin U, the compound that has an influence on the binding betweenNeuromedin U and TGR-1 can be screened.

[0194] A block of oocytes, collected from a female Xenopus Laevis numbedby ice-cooling, was treated with collagenase (0.5 mg/ml) dissolved inMBS solution (88 mM NaCl, 1 mM KCl, 0.41 mM CaCl₂, 0.33 mM Ca(NO₃)₂,0.82 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM HEPES, pH 7.4), shaking at 150 rpmfor 1-6 hours at 19° C. until the block of cells gets loose. Afterwashing for three times with MBS, TGR-1 mRNA (50 ng/50 nl) ismicroinjected into an oocyte with a micromanipulator. TGR-1 mRNA can beprepared from tissues or cells, or by in vitro transcription from aplasmid. The oocyte is cultured in MBS solution for 3 days at 20° C.,and placed in a pit of a voltage clamp devise where Ringer solutionflows. Glass microelectrodes for voltage clamp and voltmeter areinserted into the cell and the cathode is placed outside of the cell.After the potential become stable, the change in potential is recordedafter passing the Ringer solution containing Neuromedin U or NeuromedinU and a test compound. An influence of the compound that alters thebinding between Neuromedin U and TGR-1 is measured by comparing themembrane potential change of TGR-1 introduced Xenopus Laevis oocyte withthe case of single administration of Neuromedin U. The compound thatinhibits the cell membrane potential change can be selected as acandidate substance that is capable of altering the binding betweenTGR-1 and Neuromedin U. On the other hand, an agonist can be screened byadministrating only the test compound and observing the cell membranepotential change like the change observed in Neuromedin Uadministration.

[0195] In this system, poly (A)⁺ RNA of various G-protein genes can beintroduced to amplify the change so that the reaction can be measuredeasily. Also, the poly (A)⁺ RNA of protein gene, such as aequorin whichradiates light in the presence of Ca ion is injected as well so that thereaction can be measured by observing the radiation of light instead ofthe membrane potential change.

[0196] The screening kit for a compound or a salt thereof that altersthe binding property of Neuromedin U with TGR-1 comprises TGR-1, thecells containing TGR-1, or the membrane fraction of the cells containingTGR-1; and Neuromedin U.

[0197] Examples of the screening kit of the present invention are asfollows.

[0198] 1. Reagents for Screening:

[0199] (1) Buffer for Measurement and Washing

[0200] Hanks' balanced salt solution (Gibco Co.) supplemented with 0.05%bovine serum albumin (Sigma Co.).

[0201] The solution is sterilized by filtration through a 0.45 μmfilter, and stored at 4° C. or may be prepared at use.

[0202] (2) Standard TGR-1 Preparation

[0203] CHO cells expressing TGR-1 which are plated on a 12-well plate ata density of 5×10⁵ cells/well, and cultured at 37° C. under 5% CO₂ and95% air for 2 days.

[0204] (3) Labeled Ligands

[0205] Neuromedin U labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S] etc., whichis dissolved in an appropriate buffer, and stored at 4° C. or −20° C.,and diluted to 1 μM with the measurement buffer at use.

[0206] (4) Standard Ligand Solution

[0207] Neuromedin U is dissolved in PBS containing 0.1% bovine serumalbumin (Sigma Co.) at a final concentration of 1 mM, and stored at −20°C.

[0208] 2. Measurement Method:

[0209] (1) TGR-1-expressing cells are cultured in a 12-well cultureplate and washed twice with 1 ml of the measurement buffer, and 490 μlof the measurement buffer is added to each well.

[0210] (2) After adding 5 μl of 10⁻³-10⁻¹⁰ M test compound solution, andthen 5 μl of a labeled Neuromedin U, the cells are incubated at roomtemperature for an hour. To determine the amount of the non-specificbinding, 5 μl of 10⁻³ M Neuromedin U is added in place of the testcompound.

[0211] (3) The reaction solution is removed, and the wells are washed 3times with the washing buffer. The labeled Neuromedin U bound to thecells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquidscintillator A (Wako Pure Chemical Industries, Ltd.)

[0212] (4) The radioactivity is measured using a liquid scintillationcounter (Beckman Co.), and the percent maximum binding (PMB) iscalculated by the equation below.

PMB=[(B−NSB)/(B ₀ −NSB)]×100

[0213] PMB: Percent maximum binding

[0214] B: Value obtained in the presence of a test compound

[0215] NSB: Non-specific binding

[0216] B₀: Maximum binding

[0217] The compound obtained by the screening method or the screeningkit of the present invention, or a salt thereof is a compound thatalters (inhibits or enhances) the binding between Neuromedin U andTGR-1. Concretely, it is a compound having the TGR-1-mediated cellstimulating activity (so-called TGR-1 agonist) or a compound not havingthe cell stimulating activity (so-called TGR-1 antagonist). Thecompounds include peptides, proteins, non-peptide compounds, syntheticcompounds, and fermentation products. They may be novel or knowncompounds.

[0218] The evaluation method for determining whether it is the TGR-1agonist or TGR-1 antagonist mentioned above is described in (i) and (ii)below.

[0219] (i) By conducting binding assay according to the screeningmethods (1)-(3) above, the compound that alters (especially, inhibits)the binding property between Neuromedin U and TGR-1 is obtained. Then,the obtained compound is assayed as to whether it has theabove-mentioned TGR-1-mediated cell stimulating activities or not. Thecompound having the cell stimulating activities or a salt thereof isdetermined to be a TGR-1 agonist, and the compound not having the cellstimulating activities or a salt thereof is determined to be a TGR-1antagonist.

[0220] (ii)(a) The above-mentioned TGR-1-mediated cell-stimulatingactivity is measured after contacting a test compound to cells havingTGR-1. The compound having the cell stimulating activity or salt thereofis a TGR-1 agonist.

[0221] (b) The above-mentioned TGR-1-mediated cell-stimulating activityis measured and compared between when the compound that activates TGR-1(e.g. Neuromedin U or TGR-1 agonist) is brought in contact with cellshaving TGR-1 and when the compound that activates TGR-1 and the testcompound are brought in contact with cells having TGR-1. The compound ora salt thereof which may reduce the cell stimulating activity induced bythe compound that activates TGR-1 is a TGR-1 antagonist.

[0222] Since TGR-1 agonists have the same physiological activities asthat of Neuromedin U, they are useful as safe and low-toxicpharmaceuticals.

[0223] On the other hand, since TGR-1 antagonists can inhibit thephysiological activities of Neuromedin U, they are useful as safe andlow-toxic pharmaceuticals for inhibiting the receptor activities.

[0224] Since Neuromedin U or a salt thereof relates to the smooth musclecontraction, the increase in blood pressure, the regulation ofion-transportation in intestine, and the increase in ACTH and subsequentincrease in corticosterone after its hypodermic administration, it canbe used as a prophylactic and/or therapeutic agent for hypotension, anda local vasoconstrictor. Thus, among compounds obtainable by theabove-mentioned screening methods or screening kits, TGR-1 agonists canbe useful as a prophylactic and/or therapeutic agent for hypotension, alocal vasoconstrictor, and further be useful as a uterine contractionaccelerating agent for ameliorating, preventing and treating variousdiseases associated with uterine contraction insufficiency, such as weaklabor contraction, atonic bleeding, delivery of the placenta, uterineinvolution insufficiency, artificial abortion, induction of delivery,arrest of delivery, endocervical canal asthenia, inversion of theuterus, retention of the placenta and egg membrane, postpartumhemorrhage, prolapse of the muliebria, infertility, care for mother'sbody at multiple pregnancy, malpresentation, dysmenorrhea, miscarriage,endometriosis, chronic inflammatory disease of the uterus, myoma of theuterus, deformity of the uterus, adenomyosis of the uterus,uterocervical laceration, post-traumatic stress syndrome.

[0225] TGR-1 antagonists can be useful as a prophylactic and/ortherapeutic agent for hypertension, myocardial dysfunction, acute renalfailure, stress-related diseases, for example, (1) diseases ofcardiovascular system (angina pectoris, myocardial infarction,arrhythmia, etc.), (2) diseases of respiratory system (bronchial asthma,hyperpnea syndrome, etc.), (3) diseases of musculoskeletal system (e.g.chronic arthorheumatism, lumbago, migraine, tension headache, etc.), (4)other diseases (e.g. diabetes, climacteric disorder, chronic pain,decrease of immunity, etc.), diseases of digestive system (gastriculcer, ulcerative colitis, etc). Further, TGR-1 antagonists are usefulas a uterine contraction suppressing agent for ameliorating, preventingand treating various diseases associated with excessive uterinecontraction, such as too strong contraction, pseudo-contraction,prolonged pregnancy, tonic uterine contraction, fetal asphyxia, uterinerupture, endocervical canal laceration, premature delivery, myoma of theuterus, deformity of uterus, adenomyosis of the uterus, abnormalexpulsive force, chronic inflammatory disease of the uterus, care formother's body at multiple pregnancy, malpresentation, Prader-Willisyndrom, dysmenorrhea.

[0226] Moreover, since Neuromedin U and a salt thereof has the functionof controlling appetite, among compounds obtainable by theabove-mentioned screening methods or screening kits on the basis of saidfunction, TGR-1 agonists are used as anorectic agents, anti-adiposisagents, remedies for bulimia and polyphagia, and TGR-1 antagonists areused as aperitive agents.

[0227] For the salt of the compound that can be obtained according tothe above-mentioned screening method or the screening kit, for example,the pharmacologically acceptable salt is used. The examples are a saltwith an inorganic base, a salt with an organic base, a salt with aninorganic acid, a salt with an organic acid, a salt with a basic oracidic amino acid and so on.

[0228] The preferred examples of salt with an inorganic base include analkali metal salt such as sodium salt, potassium salt; alkali earthmetal salt, calcium salt and magnesium salt; and aluminum salt, ammoniumsalt, etc.

[0229] The preferred examples of salt with an organic base includetrimethylamine salt, triethylamine salt, pyridine salt, picoline salt,2,6-lutidine salt, ethanolamine salt, diethanolamine salt,triethanolamine salt, cyclohexylamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, etc.

[0230] The preferred examples of salt with an inorganic acid arehydrochloric acid salt, hydrobromic acid salt, sulfuric acid salt,phosphoric acid salt, etc.

[0231] The preferred examples of salt with an organic acid are formicacid salt, acetic acid salt, propionic acid salt, fumaric acid salt,oxalic acid salt, tartaric acid salt, maleic acid salt, citric acidsalt, succinic acid salt, malic acid salt, methanesulfonic acid salt,benzenesulfonic acid salt, benzoic acid salt, etc.

[0232] The preferred examples of salt with a basic amino acid include asalt with arginine, lysine, ornithine, etc. The preferred examples ofsalt with an acidic amino acid include a salt with aspartic acid,glutamic acid, etc.

[0233] When the compound obtainable using the screening method or thescreening kit or a salt thereof is used as the above-mentioned drug, itcan be used as follows.

[0234] When the compound obtainable using the screening method or thescreening kit or a salt thereof is used as the above-mentioned drug, itcan be prepared by publicly known methods. For example, the compound canbe used orally as tablets having sugar coating or enteric coating asnecessary, capsules, elixirs and microcapsules, or parenterally as aninjection, such as an aseptic solution or suspension with water or otherpharmaceutically acceptable. For example, these preparations can beproduced by admixing physiologically acceptable carriers, flavors,excipients, vehicles, preservatives, stabilizers and binders with thecompound or a salt thereof of the present invention in a generallyacceptable unit dose required for pharmaceutical formulation. The amountof the active ingredient in these pharmaceutical preparations isdesigned to have a suitable dose in the designated range.

[0235] The additives which can be admixed in the tablets, capsules etc.include, for example, binders such as gelatin, corn starch, tragacanth,gum arabic; excipients such as crystalline cellulose; swelling agentssuch as corn starch, gelatin and alginic acid; lubricants such asmagnesium stearate; sweeteners such as sucrose, lactose and saccharine;and flavors such as peppermint, akamono oil and cherry. When a capsuleis in a unit dosage form, liquid carriers such as fats and oils can becontained in the materials described above. The aseptic composition forinjection can be formulated according to conventional pharmaceuticalformulation by dissolving or suspending the active material andnaturally occurring vegetable oils such as sesame oil and coconut oil invehicles such as injection water.

[0236] The aqueous liquid for injection includes, for example,physiological saline or an isotonic solution containing glucose andother supplementary agents (e.g., D-sorbitol, D-mannitol, sodiumchloride etc.), and may be used in combination with suitable solubilizersuch as alcohols (e.g., ethanol etc.), polyalcohols (e.g., propyleneglycol, polyethylene glycol etc.) and nonionic surfactants (e.g.,Polysorbate 80™, HCO-50 etc.). The oily liquid includes, for example,sesame oil, soybean oil etc., and may be used in combination withsolubilizer such as benzyl benzoate, benzyl alcohol etc.

[0237] Further, it may contain buffers (e.g., phosphate buffer, sodiumacetate buffer etc.), soothing agents (e.g., benzalkonium chloride,procaine hydrochloride etc.), stabilizers (e.g., human serum albumin,polyethylene glycol etc.), preservatives (e.g., benzyl alcohol, phenoletc.), antioxidants etc. Usually, the prepared injection is filled intosuitable ampoules.

[0238] The pharmaceutical preparation thus obtained is safe and lowtoxic so that it can be administered to warm-blooded animals (e.g.,human, guinea pig, rat, mouse, pig, sheep, cow, monkey, dog andchicken), amphibian (e.g. frog) and fish.

[0239] Dose of the compound or a salt thereof (especially antagonists)which is obtained by the screening method or the screening kit of thepresent invention will vary depending on conditions. In oraladministration to an adult patient (60 kg body weight) withhypertension, the dose is normally about 0.1 to 1000 mg, preferablyabout 1.0 to 300 mg, and more preferably about 3.0 to 50 mg per day. Inparenteral administration, the single dose will also vary depending onsubject to be administered, conditions, routes for administration, etc.For example, in an injection form for an adult patient with hypertension(60 kg body weight), advantageously, a daily dose of about 0.01 to 30mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10mg will be administered intravenously. For other animal species, thedose to be administered can also be calculated according to its bodyweight from the dose for 60 kg body weight.

[0240] The present invention further relates to an antibody to TGR-1.

[0241] The antibody to TGR-1 may be any polyclonal or monoclonalantibody which is capable of recognizing TGR-1.

[0242] The antibody to TGR-1 may be produced by a well-known method forproducing an antibody or antisera, using as TGR-1 the antigen.

[0243] [Preparation of Monoclonal Antibody]

[0244] (a) Preparation of Monoclonal Antibody-Producing Cells

[0245] TGR-1 is administered to warm-blooded animals either solely ortogether with carriers or diluents to the site where the production ofantibody is possible by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually carried out once every two to six weeks andtwo to ten times in total. Examples of the applicable warm-bloodedanimals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep andgoats, with the use of mice and rats being preferred.

[0246] In the preparation of monoclonal antibody-producing cells,warm-blooded animals, e.g. mice are immunized with an antigen, aindividual having detectable antibody titer is selected, then spleen orlymph node is collected after two to five days after the finalimmunization, and antibody-producing cells contained therein are fusedwith myeloma cells to give monoclonal antibody-producing hybridomas.Measurement of the antibody titer in antisera may be carried out, forexample, by reacting a labeled TGR-1 with the antiserum, followed byassaying the binding activity of the label bound to the antibody. Thefusion may be carried out, for example, by the known method by Koehlerand Milstein (Nature, 256, 495, 1975). Examples of the fusionaccelerator are polyethylene glycol (PEG), Sendai virus, etc., of whichPEG is preferably employed.

[0247] Examples of the myeloma cells are NS-1, P3U1, SP2/0, AP-1, etc.P3U1 is preferably employed. A preferred ratio of the number of theantibody-producing cells used (spleen cells) to the number of myelomacells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by incubating at 20 to 40° C.,preferably at 30 to 37° C. for 1 to 10 minutes, an efficient cell fusioncan be carried out.

[0248] Various methods can be used for screening a monoclonalantibody-producing hybridoma. Examples of such methods include a methodwhich comprises adding the supernatant of hybridoma to a solid phase(e.g., microplate) adsorbing TGR-1 antigen directly or together with acarrier, adding an anti-immunoglobulin antibody (where mouse cells areused for the cell fusion, anti-mouse immunoglobulin antibody is used)labeled with a radioactive substance or an enzyme or Protein A, anddetecting the monoclonal antibody bound to the solid phase; and a methodwhich comprises adding the supernatant of hybridoma to a solid phaseadsorbing an anti-immunoglobulin antibody or Protein A, adding TGR-1labeled with a radioactive substance or an enzyme, and detecting themonoclonal antibody bound to the solid phase.

[0249] The monoclonal antibody can be selected according to publiclyknown methods or their modifications. In general, the selection can beeffected in a medium for animal cell supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any selection and growthmedium can be employed as far as the hybridoma can grow there. Forexample, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1% to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike can be used. The cultivation is carried out generally at 20° C. to40° C., preferably at 37° C., for about 5 days to about 3 weeks,preferably 1 to 2 weeks, normally in 5% CO₂. The antibody titer of theculture supernatant of a hybridoma can be determined as in the assay forthe antibody titer in antisera described above.

[0250] (b) Purification of Monoclonal Antibody

[0251] Separation and purification of a monoclonal antibody can becarried out in the same way as the case of a polyclonal antibody, byconventional methods for separation and purification of immunoglobulins(for example, salting-out, alcohol precipitation, isoelectric pointprecipitation, electrophoresis, adsorption and desorption with ionexchangers (e.g. DEAE), ultracentrifugation, gel filtration, or aaffinity purification method which comprises collecting only an antibodywith an activated adsorbent such as an antigen-binding solid phase,Protein A or Protein G, and dissociating the binding to obtain theantibody.

[0252] [Preparation of Polyclonal Antibody]

[0253] The polyclonal antibody of the present invention can be producedby publicly known methods or modifications thereof. For example, awarm-blooded animal is immunized with the complex of the immunogen(TGR-1) and a carrier protein in a manner similar to the methoddescribed above for the production of monoclonal antibodies. Thefraction containing the antibody to TGR-1 is collected from theimmunized animal to carry out separation and purification of theantibody.

[0254] In the complex of immunogen and carrier protein used to immunizea warm-blooded animal, any type of carrier protein may be crosslinked tothe hapten in any mixing ratio of carrier to hapten, as long as theantibody is efficiently produced to the immunized complex. For example,bovine serum albumin, bovine thyroglobulin or keyhole limpet hemocyanin,etc. is coupled to hapten in a carrier-to-hapten weight ratio ofapproximately 0.1 to 20, preferably about 1 to about 5.

[0255] A variety of condensation agents can be used for the coupling ofcarrier to hapten. Glutaraldehyde, carbodiimide, maleimide activatedester and activated ester reagents containing thiol group ordithiopyridyl group are used for the coupling.

[0256] The condensation product is administered to warm-blooded animalseither solely or together with carriers or diluents to the site that canproduce the antibody by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once every 2 to 6 weeks and 3 to 10 timesin total.

[0257] The polyclonal antibody can be collected from the blood, ascites,etc., preferably from the blood of warm-blooded animal immunized by themethod described above.

[0258] The titer of polyclonal antibody in antiserum can be assayed bythe same procedure as that for the determination of titer of serumantibody described above. The separation and purification of thepolyclonal antibody can be carried out according to the method for theseparation and purification of immunoglobulins as performed in theseparation and purification of monoclonal antibodies described above.

[0259] The antibody of the present invention is capable of recognizingspecifically TGR-1. Therefore, the antibody can be used to quantifyTGR-1 in a test fluid, especially by the sandwich immunoassay, etc.Thus, the present invention provides, for example, the followingquantification methods:

[0260] (i) a method of quantifying TGR-1 in a test fluid, whichcomprises reacting the antibody of the present invention competitivelywith the test fluid and the labeled TGR-1; and measuring the ratio ofthe labeled TGR-1 bound to the antibody; and,

[0261] (ii) a method of quantifying TGR-1 in a test fluid, whichcomprises reacting the test fluid with the antibody of the presentinvention immobilized on a carrier and the labeled antibody of thepresent invention simultaneously or sequentially; and measuring theactivity of the label on the immobilizing carrier.

[0262] In (ii) described above, it is preferred that one antibodyrecognizes the N-terminal region of TGR-1, and the other antibody reactswith the C-terminal region of TGR-1.

[0263] Using the monoclonal antibody to TGR-l (hereinafter sometimesreferred to as the monoclonal antibody of the present invention), TGR-1can be assayed and also detected by tissue staining. For this purpose,an antibody molecule itself may be used, or F(ab′)₂, Fab′ or Fabfractions of the antibody molecule may also be used. Assay methods usingthe antibody to TGR-1 are not particularly limited. Any assay method canbe used, so long as the amount of antibody, antigen, or antibody-antigencomplex corresponding to the amount of antigen (e.g., the amount ofTGR-1) in the test fluid can be detected by chemical or physical meansand the amount of the antigen can be calculated from a standard curveprepared from standard solutions containing known amounts of theantigen. For example, nephrometry, competitive methods, immunometricmethod, and sandwich method are appropriately used, with the sandwichmethod described below being most preferable in terms of sensitivity andspecificity.

[0264] As the labeling agent for the methods using labeled substances,there are employed, for example, radioisotopes, enzymes, fluorescentsubstances, luminescent substances, etc. For the radioisotope, forexample, [¹²⁵I], [¹³¹I], [³H] and [¹⁴C] are used. As the enzymedescribed above, stable enzymes with high specific activity arepreferred; for example, β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, malate dehydrogenase and the like are used.Examples of the fluorescent substance used are fluorescamine andfluorescein isothiocyanate. For the luminescent substance, for example,luminol, luminol derivatives, luciferin, and lucigenin. Furthermore, thebiotin-avidin system may be used for binding antibody or antigen to thelabel.

[0265] For immobilization of antigen or antibody, physical adsorptionmay be used. Chemical binding methods conventionally used forinsolubilization or immobilization of proteins or enzymes may also beused. For the carrier, for example, insoluble polysaccharides such asagarose, dextran, cellulose, etc.; synthetic resin such as polystyrene,polyacrylamide, silicon, etc., and glass or the like are used.

[0266] In the sandwich method, the immobilized monoclonal antibody ofthe present invention is reacted with a test fluid (primary reaction),then with the labeled monoclonal antibody of the present invention(secondary reaction), and the activity of the label on the immobilizingcarrier is measured, whereby the amount of TGR-1 in the test fluid canbe quantified. The order of the primary and secondary reactions may bereversed, and the reactions may be performed simultaneously or with aninterval. The labeling agent and method for immobilization may be thesame as those described above.

[0267] In the sandwich immunoassay, the immobilized or labeled antibodyis not necessarily based on one type of antibody, but a mixture of twoor more types of antibodies may be used to improve assay sensitivity orthe like.

[0268] In the sandwich assay for measuring TGR-1, the monoclonalantibodies of the present invention which bind to different sites ofTGR-1 are preferably used in the first and second reactions. Thus, as tothe antibodies used in the first and second reactions, for example, whenthe antibody used in the second reaction recognizes the C-terminalregion of TGR-1, it is preferable to use the antibody recognizingregions other than the C-terminal region, e.g., the N-terminal region inthe first reaction.

[0269] The monoclonal antibody of the present invention can be used forassay systems other than the sandwich method, for example, thecompetitive method, immunometric method, nephrometry, etc. In thecompetitive method, antigen in a test fluid and the labeled antigen arecompetitively reacted with antibody, and the unreacted labeled antigen(F) and the labeled antigen bound to the antibody (B) are separated (B/Fseparation). By measuring the amount of the label in B or F, the amountof the antigen in the test fluid is determined. The above reactionmethod includes a liquid phase method using an antibody in a solubleform, polyethylene glycol for B/F separation and a secondary antibody tothe soluble antibody; and an solid phase method either using theimmobilized primary antibody, or using the soluble primary antibody andthe immobilized secondary antibody.

[0270] In the immunometric method, after reacting antigen in a testfluid and immobilized antigen competitively with a definite amount oflabeled antibody, the solid phase is separated from the liquid phase.Alternatively, after reacting antigen in a test fluid with an excessamount of labeled antibody, and then adding immobilized antigen toadsorb the unreacted labeled antibody, the solid phase is separated fromthe liquid phase. Then, the amount of the label in either phase ismeasured to quantify the antigen in the test fluid.

[0271] In the nephrometry, an insoluble precipitate produced after theantigen-antibody reaction in gel or solution is quantified. When theamount of antigen in the test fluid is so small that only a small amountof precipitate is obtained, laser nephrometry using scattering of laseris advantageously employed.

[0272] For applying these immunological methods to the measuring methodsof the present invention, any particular conditions or procedures arenot required. Systems for measuring the rat TGR-1 of the presentinvention or its salts are constructed by adding the usual technicalconsideration in the art to the conventional conditions and procedures.For the details of these general technical means, reference can be madeto the following reviews and texts. See, for example, Hiroshi Irie, ed.“Radioimmunoassay” (Kodansha, published in 1974); Hiroshi Irie, ed.“Sequel to the Radioimmunoassay” (Kodansha, published in 1979); EijiIshikawa, et al. ed. “Enzyme immonoassay” (Igakushoin, published in1978); Eiji Ishikawa, et al. ed. “Immunoenzyme assay” (2nd ed.)(Igakushoin, published in 1982); Eiji Ishikawa, et al. ed. “Immunoenzymeassay” (3rd ed.) (Igakushoin, published in 1987); Methods in ENZYMOLOGY,Vol. 70 (Immunochemical Techniques (Part A)); ibid., Vol. 73(Immunochemical Techniques (Part B)); ibid., Vol. 74 (ImmunochemicalTechniques (Part C)); ibid., Vol. 84 (Immunochemical Techniques (Part D:Selected Immunoassays)); ibid., Vol. 92 (Immunochemical Techniques (PartE: Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol.121 (Immunochemical Techniques (Part I: Hybridoma Technology andMonoclonal Antibodies))(all published by Academic Press Publishing).

[0273] In this way, TGR-1 can be quantified in high sensitivity usingthe antibody of the present invention.

[0274] Further, it is possible to conduct diagnosis on various diseaseswhich are associated with dysfunction of TGR-1 through quantification ofTGR-1 in vivo using the antibody of the present invention.

[0275] The antibody of the present invention can also be used forspecific detection of TGR-1 present in test samples, such as body fluidsor tissues. The antibody may also be used for preparation of antibodycolumns for purification of TGR-1, for detection of TGR-1 in fractionsupon purification, and for analysis of the behavior of TGR-1 in the testcells.

[0276] In the specification and drawings, nucleic acids and amino acidsare denoted in accordance with the IUPAC-IUB Commission on BiochemicalNomenclature or the conventional manner in the art, examples of whichare shown below. The optical isomer of amino acid is L form unlessotherwise indicated.

[0277] DNA: deoxyribonucleic acid

[0278] cDNA: complementary deoxyribonucleic acid

[0279] A: adenine

[0280] T: thymine

[0281] G: guanine

[0282] C: cytosine

[0283] Y: thymine or cytosine

[0284] N: adenine, guanine, cytosine or thymine

[0285] R: adenine or guanine

[0286] M: adenine or cytosine

[0287] W: adenine or thymine

[0288] S: guanine or cytosine

[0289] RNA: ribonucleic acid

[0290] mRNA: messenger ribonucleic acid

[0291] dATP: deoxyadenosine triphosphate

[0292] dTTP: deoxythymidine triphosphate

[0293] dGTP: deoxyguanosine triphosphate

[0294] dCTP: deoxycytidine triphosphate

[0295] ATP: adenosine triphosphate

[0296] EDTA: ethylenediaminetetraacetic acid

[0297] SDS: sodium dodecyl sulfate

[0298] TFA: trifluoroacetic acid

[0299] EIA: enzyme immunoassay

[0300] Gly or G: glycine

[0301] Ala or A: alanine

[0302] Val or V: valine

[0303] Leu or L: leucine

[0304] Ile or I: isoleucine

[0305] Ser or S: serine

[0306] Thr or T: threonine

[0307] Cys or C: cysteine

[0308] Met or M: methionine

[0309] Glu or E: glutamic acid

[0310] Asp or D: aspartic acid

[0311] Lys or K: lysine

[0312] Arg or R: arginine

[0313] His or H: histidine

[0314] Phe or F: phenylalanine

[0315] Tyr or Y: tyrosine

[0316] Trp or W: tryptophan

[0317] Pro or P: proline

[0318] Asn or N: asparagine

[0319] Gln or Q: glutamine

[0320] pGlu: pyroglutamic acid

[0321] Me: methyl

[0322] Et: ethyl

[0323] Bu: butyl

[0324] Ph: phenyl

[0325] TC: thiazolidine-4(R)-carboxamide

[0326] Bom: benzyloxymethyl

[0327] NMP: N-methylpyrrolidone

[0328] PAM: phenylacetoamidemethyl

[0329] The substituents, protective groups and reagents, which arefrequently used in the specification, are shown by the followingabbreviations.

[0330] Tos: p-toluenesulfonyl

[0331] HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide

[0332] Bzl: benzyl

[0333] Z: benzyloxycarbonyl

[0334] Br-Z: 2-bromobenzyloxycarbonyl

[0335] Cl-Z: 2-chlorobenzyloxycarbonyl

[0336] Boc: t-butoxycarbonyl

[0337] HOBt: 1-hydroxybenztriazole

[0338] DCC: N,N′-dicyclohexylcarbodiimide

[0339] TFA: trifluoroacetic acid

[0340] Fmoc: N-9-fluorenylmethoxycarbonyl

[0341] DNP: dinitrophenyl

[0342] Bum: t-butoxymethyl

[0343] Trt: trityl

[0344] BSA: bovine serum albumin

[0345] CHAPS: 3-[(3-colamidepropyl)dimethylanmmonio]-1-propane sulfonate

[0346] PMSF: phenylmethylsulfonylfluoride

[0347] E64: (L-3-trans-caroboxoirane-2-carbonyl) L-leucyl-agumatin

[0348] GDP: Guanosine-5′-diphosphate

[0349] MEM á: minimum essential medium alpha

[0350] Fura-2AM:1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5methylphenoxy)-ethane-N′,N′,N′,N′-tetra acetic acid-pentacetoxymethylester

[0351] HBSS: Hanks' Balanced Salt Solution

[0352] Fluo-3AM:1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N′,N′,N′,N′-tetraacetic acid pentaacetoxymethyl ester

[0353] HEPES: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid

[0354] MeBzl: 4-methylbenzyl

[0355] NMP: N-methylpyrrolidone

[0356] Each of the sequence identification numbers in the SequenceListing indicates the following sequence.

[0357] [SEQ ID NO:1]

[0358] This shows the amino acid sequence of TGR-1 obtained in Example1.

[0359] [SEQ ID NO:2]

[0360] This shows the nucleic acid sequence of DNA encoding TGR-1 havingthe amino acid sequence shown by SEQ ID NO:1.

[0361] [SEQ ID NO:3]

[0362] This shows the nucleic acid sequence of primer 1 described inExample 1.

[0363] [SEQ ID NO:4]

[0364] This shows the nucleic acid sequence of primer 2 described inExample 1.

[0365] [SEQ ID NO:5]

[0366] This shows the amino acid sequence of pig Neuromedin U-8.

[0367] [SEQ ID NO:6]

[0368] This shows the amino acid sequence of dog Neuromedin U-8.

[0369] [SEQ ID NO:7]

[0370] This shows the amino acid sequence of chicken Neuromedin U-9.

[0371] [SEQ ID NO:8]

[0372] This shows the amino acid sequence of guinea pig Neuromedin U-9.

[0373] [SEQ ID NO:9]

[0374] This shows the amino acid sequence of rat Neuromedin U-23.

[0375] [SEQ ID NO:10]

[0376] This shows the amino acid sequence of frog Neuromedin U-23.

[0377] [SEQ ID NO:11]

[0378] This shows the amino acid sequence of human Neuromedin U-25.

[0379] [SEQ ID NO:12]

[0380] This shows the amino acid sequence of pig Neuromedin U-25.

[0381] [SEQ ID NO:13]

[0382] This shows the amino acid sequence of dog Neuromedin U-25.

[0383] [SEQ ID NO:14]

[0384] This shows the amino acid sequence of chicken Neuromedin U-25.

[0385] [SEQ ID NO:15]

[0386] This shows the amino acid sequence of frog Neuromedin U-25.

[0387] [SEQ ID NO:16]

[0388] This shows an amino acid sequence of a partial peptide ofNeuromedin U-25. This corresponds to an amino acid sequence at the 4 to8 positions in the amino acid sequence shown by SEQ ID NO: 5.

[0389] [SEQ ID NO:17]

[0390] This shows the amino acid sequence which is substantially thesame sequence as TGR-1 (as described in WO 99/55732).

[0391] [SEQ ID NO:18]

[0392] This shows the nucleic acid sequence of DNA encoding the aminoacid sequence shown by SEQ ID NO:23.

[0393] [SEQ ID NO:19]

[0394] This shows the nucleic acid sequence of the primer RTGRF2described in Example 3.

[0395] [SEQ ID NO:20]

[0396] This shows the nucleic acid sequence of the primer RTGRR1described in Example 3.

[0397] [SEQ ID NO:21]

[0398] This shows the amino acid sequence of rat TGR-1 obtained inExample 3.

[0399] [SEQ ID NO:22]

[0400] This shows the nucleic acid sequence of DNA encoding rat TGR-1.

[0401]Escherichia coli transformant TOP10/pCR2.1TOPO-TGR-1 which hascDNA encoding TGR-1 shown by SEQ ID NO:1 obtained in Example 1 was ondeposit with the Ministry of International Trade and Industry, Agency ofIndustrial Science and Technology, National Institute of Bioscience andHuman Technology (NIBH), located at 1-1-3, Higashi, Tsukuba-shi,Ibaraki, Japan, as the Accession Number FERM BP-6964 on Dec. 6, 1999;and with Institute for Fermentation (IFO), located at 2-17-85, JusoHoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, as the Accession NumberIFO 16336 on Nov. 12, 1999.

[0402]Escherichia coli transformant JM109/prTGR-1 which has cDNAencoding TGR-1 shown by SEQ ID NO:21 obtained in Example 3 was ondeposit with the Ministry of International Trade and Industry, Agency ofIndustrial Science and Technology, National Institute of Bioscience andHuman Technology (NIBH), located at 1-1-3, Higashi, Tsukuba-shi,Ibaraki, Japan, as the Accession Number FERM BP-7355 on Nov. 9, 2000;and with Institute for Fermentation (IFO), located at 2-17-85, JusoHoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, as the Accession NumberIFO 16488 on Oct. 24, 2000.

EXAMPLES

[0403] The following examples are intended to illustrate the presentinvention in detail, but not intended to limit the scope of the presentinvention.

Example 1

[0404] Cloning of cDNA Encoding TGR-1 and Determination of its NucleicAcid Sequence

[0405] A PCR was carried out using human testis cDNA (Marathon-Ready™cDNA; Clontech) as a template, and 2 primers, primer 1 (SEQ ID NO:3) andprimer 2 (SEQ ID NO:4). The PCR, using Advantage 2 Polymerase Mixture(Clontech), followed (i) 95° C. for 1 min.; (ii) 5 cycles of 95° C. for30 sec. and 68° C. for 2 min.; (iii) 5 cycles of 95° C. for 30 sec., 64°C. for 30 sec. and 68° C. for 2 min.; (iv) 30 cycles of 95° C. for 30sec., 62° C. for 30 sec. and 68° C. for 2 min.; and (v) 68° C. for 7min. for elongation. After the reaction, the reaction product wassubcloned into a plasmid vector pCR2.1TOPO according to the instructionfor TA cloning Kit (Invitrogen). The resulting plasmid was introducedinto E. coli TOP10, and a clone having the plasmid was selected in a LBagar plate containing ampicilin. As a result of analysis of each clonalsequence, cDNA sequence (SEQ ID NO:2) encoding TGR-1 (SEQ ID NO:1) wasobtained.

Example 2

[0406] Comparison of Neuromedin U-8-Induced Responses inTGR-1-Expressing CHO Cells and Mock CHO Cells by a Site Sensor Technique

[0407] TGR-1-expressing CHO cells were prepared according to a knownmethod using the cDNA encoding TGR-1, obtained in Example 1.TGR-1-expressing CHO cells and mock CHO cells were plated in thecapsules for site sensor at a density of 2.7×10⁵ cells/well, andcultured overnight. The capsules containing the cells were set in thesite sensor, and these cells were refluxed with a low-buffered RMPImedium supplemented with 0.1% bovine serum albumin for acclimation.Repeating a pumping cycle of ON (80 seconds) and OFF (40 seconds), arate of change in extracellular pH was measured as an acidification ratewith the site sensor with time of pumping off.

[0408] Pig Neuromedin U-8 (BACHEM, H-5505, SEQ ID NO:5) was dissolved inthe medium, and the diluted solutions thereof were prepared at thestep-wise decreased concentrations. They were exposed to the cells for 7minutes and 2 seconds through switching the flow paths. As a result ofcomparing the peak reaction values after corrected setting the valuesobtained during 3 cycles immediately before the exposure of the dilutionto the cells as 100%, it was observed that TGR-1-expressing CHO cellsreacted to Neuromedin U in a specific and does-dependent manner (FIG. 1)

Example 3

[0409] Obtaining cDNA encoding Rat TGR-1

[0410] To obtain a fragment of a full-length cDNA encoding Rat TGR-1,the following 2 kinds of DNAs were synthesized.

[0411] RTGRF2:5′-CTGATGCTATCCTTTCACTCTCTCAGACC-3′ (SEQ ID NO:19)

[0412] RTGRR1:5′-TCCTTGCAGTTTTGGCACATAGATGGA-3′ (SEQ ID NO:20)

[0413] Using these synthetic DNAs, RTGRF2 and RTGRR1 as primers, andcDNAs synthesized from poly(A) ⁺RNA of rat uterus as a template, a PCRwas carried out to amplify a fragment encoding the full-length cDNA. Thereaction solution for the PCR was a total volume 25 μl composed of 2 μlcDNA solution (derived from 8 ng poly(A)⁺ RNA), 1 μl dNTP (10 mM), 0.5μl Advantage 2 DNA polymerase (Clonetech), 2.5 μl 10× buffer solutionattached to the DNA polymerase product, 18 μl distilled water, and 0.5μl of each RTGRF2 and RTGRR1 (each 10 μM). After heating the reactionsolution at 95° C. for 2 minutes for denaturation, the PCR was carriedout by repeating 31 times a cycle of 98° C. for 10 seconds and 65° C.for 90 seconds. About 1.4 kb PCR product was detected usingelectrophoresis, purified using QIA quick Gel Extraction Kit (Quiagen),inserted into the cloning vector pCR2.1TOPO according to the instructionfor TA cloning Kit (Invitrogen), and introduced into E. coli JM109 togive a transformant E. coli JM109/prTGR-1. The nucleic acid sequenceinserted in the plasmid prTGR-1 was determined (SEQ ID NO:22), and theamino acid sequence predicted from the coding sequence was shown in SEQID NO:21. In addition, FIG. 2 shows comparison of this amino acidsequence with the human sequence (SEQ ID NO:1) obtained in Example 1.

INDUSTRIAL APPLICABILITY

[0414] The screening method for a compound or a salt thereof that altersthe binding property between Neuromedin U and TGR-1, characterized byusing Neuromedin U and TGR-1, can be useful for screening a therapeuticand/or prophylactic agent for obesity, hypertension and stress-relateddiseases. A TGR-1 agonist can be useful as a therapeutic and/orprophylactic agent for obesity, etc. A TGR-1 antagonist can be useful asa therapeutic and/or prophylactic agent for hypertension andstress-related diseases, etc.

1 22 1 415 PRT Human 1 Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser TrpIle Tyr Gln Gln 1 5 10 15 Lys Leu Glu Asp Pro Phe Gln Lys His Leu AsnSer Thr Glu Glu Tyr 20 25 30 Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser HisPhe Phe Leu Pro Val 35 40 45 Ser Val Val Tyr Val Pro Ile Phe Val Val GlyVal Ile Gly Asn Val 50 55 60 Leu Val Cys Leu Val Ile Leu Gln His Gln AlaMet Lys Thr Pro Thr 65 70 75 80 Asn Tyr Tyr Leu Phe Ser Leu Ala Val SerAsp Leu Leu Val Leu Leu 85 90 95 Leu Gly Met Pro Leu Glu Val Tyr Glu MetTrp Arg Asn Tyr Pro Phe 100 105 110 Leu Phe Gly Pro Val Gly Cys Tyr PheLys Thr Ala Leu Phe Glu Thr 115 120 125 Val Cys Phe Ala Ser Ile Leu SerIle Thr Thr Val Ser Val Glu Arg 130 135 140 Tyr Val Ala Ile Leu His ProPhe Arg Ala Lys Leu Gln Ser Thr Arg 145 150 155 160 Arg Arg Ala Leu ArgIle Leu Gly Ile Val Trp Gly Phe Ser Val Leu 165 170 175 Phe Ser Leu ProAsn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe 180 185 190 Pro Asn GlySer Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys 195 200 205 Pro MetTrp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe 210 215 220 TyrLeu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala 225 230 235240 Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala 245250 255 Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val260 265 270 Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile AspArg 275 280 285 Leu Phe Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu AlaAla Val 290 295 300 Phe Asn Leu Val His Val Val Ser Gly Val Phe Phe TyrLeu Ser Ser 305 310 315 320 Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu SerArg Arg Phe Gln Ala 325 330 335 Ala Phe Gln Asn Val Ile Ser Ser Phe HisLys Gln Trp His Ser Gln 340 345 350 His Asp Pro Gln Leu Pro Pro Ala GlnArg Asn Ile Phe Leu Thr Glu 355 360 365 Cys His Phe Val Glu Leu Thr GluAsp Ile Gly Pro Gln Phe Pro Cys 370 375 380 Gln Ser Ser Met His Asn SerHis Leu Pro Thr Ala Leu Ser Ser Glu 385 390 395 400 Gln Met Ser Arg ThrAsn Tyr Gln Ser Phe His Phe Asn Lys Thr 405 410 415 2 1245 DNA Human 2atgtcaggga tggaaaaact tcagaatgct tcctggatct accagcagaa actagaagat 60ccattccaga aacacctgaa cagcaccgag gagtatctgg ccttcctctg cggacctcgg 120cgcagccact tcttcctccc cgtgtctgtg gtgtatgtgc caatttttgt ggtgggggtc 180attggcaatg tcctggtgtg cctggtgatt ctgcagcacc aggctatgaa gacgcccacc 240aactactacc tcttcagcct ggcggtctct gacctcctgg tcctgctcct tggaatgccc 300ctggaggtct atgagatgtg gcgcaactac cctttcttgt tcgggcccgt gggctgctac 360ttcaagacgg ccctctttga gaccgtgtgc ttcgcctcca tcctcagcat caccaccgtc 420agcgtggagc gctacgtggc catcctacac ccgttccgcg ccaaactgca gagcacccgg 480cgccgggccc tcaggatcct cggcatcgtc tggggcttct ccgtgctctt ctccctgccc 540aacaccagca tccatggcat caagttccac tacttcccca atgggtccct ggtcccaggt 600tcggccacct gtacggtcat caagcccatg tggatctaca atttcatcat ccaggtcacc 660tccttcctat tctacctcct ccccatgact gtcatcagtg tcctctacta cctcatggca 720ctcagactaa agaaagacaa atctcttgag gcagatgaag ggaatgcaaa tattcaaaga 780ccctgcagaa aatcagtcaa caagatgctg tttgtcttgg tcttagtgtt tgctatctgt 840tgggccccgt tccacattga ccgactcttc ttcagctttg tggaggagtg gagtgaatcc 900ctggctgctg tgttcaacct cgtccatgtg gtgtcaggtg tcttcttcta cctgagctca 960gctgtcaacc ccattatcta taacctactg tctcgccgct tccaggcagc attccagaat 1020gtgatctctt ctttccacaa acagtggcac tcccagcatg acccacagtt gccacctgcc 1080cagcggaaca tcttcctgac agaatgccac tttgtggagc tgaccgaaga tataggtccc 1140caattcccat gtcagtcatc catgcacaac tctcacctcc caacagccct ctctagtgaa 1200cagatgtcaa gaacaaacta tcaaagcttc cactttaaca aaacc 1245 3 30 DNAArtificial Sequence primer 3 gtcgacttaa tgtcagggat ggaaaaactt 30 4 30DNA Artificial Sequence primer 4 actagttcag gttttgttaa agtggaagct 30 5 8PRT Pig 5 Tyr Phe Leu Phe Arg Pro Arg Asn 1 5 8 6 8 PRT Dog 6 Glu PheLeu Phe Arg Pro Arg Asn 1 5 8 7 9 PRT Chicken 7 Gly Tyr Phe Phe Phe ArgPro Arg Asn 1 5 9 8 9 PRT Guinea pig 8 Gly Tyr Phe Leu Phe Arg Pro ArgAsn 1 5 9 9 23 PRT Rat 9 Tyr Lys Val Asn Glu Tyr Gln Gly Pro Val Ala ProSer Gly Gly Phe 1 5 10 15 Phe Leu Phe Arg Pro Arg Asn 20 23 10 23 PRTFrog 10 Ser Asp Glu Glu Val Gln Val Pro Gly Gly Val Ile Ser Asn Gly Tyr1 5 10 15 Phe Leu Phe Arg Pro Arg Asn 20 23 11 25 PRT Human 11 Phe ArgVal Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg 1 5 10 15 GlyTyr Phe Leu Phe Arg Pro Arg Asn 20 25 12 25 PRT Pig 12 Phe Leu Val AspGlu Glu Phe Gln Gly Pro Ile Val Ser Gln Asn Arg 1 5 10 15 Arg Tyr PheLeu Phe Arg Pro Arg Asn 20 25 13 25 PRT Dog 13 Phe Arg Leu Asp Glu GluPhe Gln Gly Pro Ile Ala Ser Gln Val Arg 1 5 10 15 Arg Gln Phe Leu PheArg Pro Arg Asn 20 25 14 25 PRT Chicken 14 Tyr Lys Val Asp Glu Asp LeuGln Gly Ala Gly Gly Ile Gln Ser Arg 1 5 10 15 Gly Tyr Phe Phe Phe ArgPro Arg Asn 20 25 15 25 PRT Frog 15 Leu Lys Pro Asp Glu Glu Leu Gln GlyPro Gly Gly Val Leu Ser Arg 1 5 10 15 Gly Tyr Phe Val Phe Arg Pro ArgAsn 20 25 16 5 PRT Human 16 Phe Arg Pro Arg Asn 1 5 17 415 PRT Human 17Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln 1 5 1015 Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr 20 2530 Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val 35 4045 Ser Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val 50 5560 Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr 65 7075 80 Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu 8590 95 Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe100 105 110 Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe GluThr 115 120 125 Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser ValGlu Arg 130 135 140 Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu GlnSer Thr Arg 145 150 155 160 Arg Arg Ala Leu Arg Ile Leu Gly Ile Val TrpGly Phe Ser Val Leu 165 170 175 Phe Ser Leu Pro Asn Thr Ser Ile His GlyIle Lys Phe His Tyr Phe 180 185 190 Pro Asn Gly Ser Leu Val Pro Gly SerAla Thr Cys Thr Val Ile Lys 195 200 205 Pro Met Trp Ile Tyr Asn Phe IleIle Gln Val Thr Ser Phe Leu Phe 210 215 220 Tyr Leu Leu Pro Met Thr ValIle Ser Val Leu Tyr Tyr Leu Met Ala 225 230 235 240 Leu Arg Leu Lys LysAsp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala 245 250 255 Asn Ile Gln ArgPro Cys Arg Lys Ser Val Asn Lys Met Leu Leu Val 260 265 270 Leu Val LeuVal Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg 275 280 285 Leu PhePhe Ser Phe Val Glu Glu Trp Thr Glu Ser Leu Ala Ala Val 290 295 300 PheAsn Leu Val His Val Val Ser Gly Val Leu Phe Tyr Leu Ser Ser 305 310 315320 Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala 325330 335 Ala Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln340 345 350 His Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu ThrGlu 355 360 365 Cys His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln PhePro Cys 370 375 380 Gln Ser Ser Val His Asn Ser His Leu Pro Thr Ala LeuSer Ser Glu 385 390 395 400 Gln Met Ser Arg Thr Asn Tyr Gln Ser Phe HisPhe Asn Lys Thr 405 410 415 18 1245 DNA Human 18 atgtcaggga tggaaaaacttcagaatgct tcctggatct accagcagaa actagaagat 60 ccattccaga aacacctgaacagcaccgag gagtatctgg ccttcctctg cggacctcgg 120 cgcagccact tcttcctccccgtgtctgtg gtgtatgtgc caatttttgt ggtgggggtc 180 attggcaatg tcctggtgtgcctggtgatt ctgcagcacc aggctatgaa gacgcccacc 240 aactactacc tcttcagcctggcggtctct gacctcctgg tcctgctcct tggaatgccc 300 ctggaggtct atgagatgtggcgcaactac cctttcttgt tcgggcccgt gggctgctac 360 ttcaagacgg ccctctttgagaccgtgtgc ttcgcctcca tcctcagcat caccaccgtc 420 agcgtggagc gctacgtggccatcctacac ccgttccgcg ccaaactgca gagcacccgg 480 cgccgggccc tcaggatcctcggcatcgtc tggggcttct ccgtgctctt ctccctgccc 540 aacaccagca tccatggcatcaagttccac tacttcccca atgggtccct ggtcccaggt 600 tcggccacct gtacggtcatcaagcccatg tggatctaca atttcatcat ccaggtcacc 660 tccttcctat tctacctcctccccatgact gtcatcagtg tcctctacta cctcatggca 720 ctcagactaa agaaagacaaatctcttgag gcagatgaag ggaatgcaaa tattcaaaga 780 ccctgcagaa aatcagtcaacaagatgctg cttgtcttgg tcttagtgtt tgctatctgt 840 tgggccccgt tccacattgaccgactcttc ttcagctttg tggaggagtg gactgaatcc 900 ctggctgctg tgttcaacctcgtccatgtg gtgtcaggtg tcttattcta cctgagctca 960 gctgtcaacc ccattatctataacctactg tctcgccgct tccaggcagc attccagaat 1020 gtgatctctt ctttccacaaacagtggcac tcccagcatg acccacagtt gccacctgcc 1080 cagcggaaca tcttcctgacagaatgccac tttgtggagc tgaccgaaga tataggtccc 1140 caattcccat gtcagtcatccgtgcacaac tctcacctcc caacagccct ctctagtgaa 1200 cagatgtcaa gaacaaactatcaaagcttc cactttaaca aaacc 1245 19 29 DNA Artificial Sequence primer 19ctgatgctat cctttcactc tctcagacc 29 20 27 DNA Artificial Sequence primer20 tccttgcagt tttggcacat agatgga 27 21 395 PRT Rat 21 Met Gly Lys LeuGlu Asn Ala Ser Trp Ile His Asp Pro Leu Met Lys 1 5 10 15 Tyr Leu AsnSer Thr Glu Glu Tyr Leu Ala His Leu Cys Gly Pro Lys 20 25 30 Arg Ser AspLeu Ser Leu Pro Val Ser Val Ala Tyr Ala Leu Ile Phe 35 40 45 Leu Val GlyVal Met Gly Asn Leu Leu Val Cys Met Val Ile Val Arg 50 55 60 His Gln ThrLeu Lys Thr Pro Thr Asn Tyr Tyr Leu Phe Ser Leu Ala 65 70 75 80 Val SerAsp Leu Leu Val Leu Leu Leu Gly Met Pro Leu Glu Ile Tyr 85 90 95 Glu MetTrp His Asn Tyr Pro Phe Leu Phe Gly Pro Val Gly Cys Tyr 100 105 110 PheLys Thr Ala Leu Phe Glu Thr Val Cys Phe Ala Ser Ile Leu Ser 115 120 125Val Thr Thr Val Ser Val Glu Arg Tyr Val Ala Ile Val His Pro Phe 130 135140 Arg Ala Lys Leu Glu Ser Thr Arg Arg Arg Ala Leu Arg Ile Leu Ser 145150 155 160 Leu Val Trp Ser Phe Ser Val Val Phe Ser Leu Pro Asn Thr SerIle 165 170 175 His Gly Ile Lys Phe Gln His Phe Pro Asn Gly Ser Ser ValPro Gly 180 185 190 Ser Ala Thr Cys Thr Val Thr Lys Pro Met Trp Val TyrAsn Leu Ile 195 200 205 Ile Gln Ala Thr Ser Phe Leu Phe Tyr Ile Leu ProMet Thr Leu Ile 210 215 220 Ser Val Leu Tyr Tyr Leu Met Gly Leu Arg LeuLys Arg Asp Glu Ser 225 230 235 240 Leu Glu Ala Asn Lys Val Ala Val AsnIle His Arg Pro Ser Arg Lys 245 250 255 Ser Val Thr Lys Met Leu Phe ValLeu Val Leu Val Phe Ala Ile Cys 260 265 270 Trp Thr Pro Phe His Val AspArg Leu Phe Phe Ser Phe Val Glu Glu 275 280 285 Trp Thr Glu Ser Leu AlaAla Val Phe Asn Leu Ile His Val Val Ser 290 295 300 Gly Val Phe Phe TyrLeu Ser Ser Ala Val Asn Pro Ile Ile Tyr Asn 305 310 315 320 Leu Leu SerArg Arg Phe Arg Ala Ala Phe Arg Asn Val Val Ser Pro 325 330 335 Thr CysLys Trp Cys His Pro Arg His Gln Pro Gln Gly Pro Pro Ala 340 345 350 GlnLys Ile Ile Phe Leu Thr Glu Cys His Leu Met Glu Leu Thr Glu 355 360 365Asp Ala Gly Pro Gln Phe Pro Gly Gln Ser Ser Ile His Asn Thr Asn 370 375380 Leu Thr Met Ala Pro Cys Ala Gly Glu Val Pro 385 390 395 22 1185 DNARat 22 atgggaaaac ttgaaaatgc ttcctggatc cacgatccac tcatgaagta cttgaacagc60 acagaggagt acttggccca cctgtgtgga cccaagcgca gtgacctatc ccttccggtg 120tctgtggcct atgcgctgat cttcctggtg ggggtaatgg gcaatcttct ggtgtgcatg 180gtgattgtcc gacatcagac tttgaagaca cccaccaact actatctctt cagcttggca 240gtctcagatc tgctggtcct gctcttgggg atgcctctgg aaatctacga gatgtggcac 300aattaccctt tcctgttcgg gcctgtggga tgctacttca agacagccct cttcgagact 360gtgtgctttg cctccattct cagtgtcacc acggttagcg tagagcgcta tgtggccatt 420gtccaccctt tccgagccaa gctggagagc acgcggcgac gggccctcag gatcctcagc 480ctagtctgga gcttctctgt ggtcttttct ttgcccaata ccagcatcca tggcatcaag 540ttccagcact ttcccaacgg gtcctccgta cctggctcag ccacctgcac agtcaccaaa 600cccatgtggg tgtataactt gatcatccaa gctaccagct tcctcttcta catcctccca 660atgaccctca tcagcgtcct ctactacctc atggggctca ggctgaagag agatgaatcc 720cttgaggcga acaaagtggc tgtgaatatt cacagaccct ctagaaagtc agtcaccaag 780atgctgtttg tcttggtcct cgtgtttgcc atctgctgga cccccttcca tgtggaccgg 840ctcttcttca gctttgtgga agagtggaca gagtccctgg ctgctgtgtt caacctcatc 900catgtggtat caggtgtctt cttttatctg agctccgcgg tcaaccccat tatctataac 960ctcctgtctc ggcgcttccg ggcggccttt cgaaatgttg tctcccctac ctgcaaatgg 1020tgccatcccc ggcatcagcc acagggacct ccagcccaga agatcatctt cttgacagaa 1080tgtcacctca tggagctgac agaggatgca ggcccccagt tccctggtca gtcatccatc 1140cacaacacca accttaccat ggccccctgt gcgggagagg tacca 1185

1. A method for screening a compound or a salt thereof that alters thebinding property of Neuromedin U or a salt thereof with a protein or asalt thereof comprising the same or substantially the same amino acidsequence as the sequence shown by SEQ ID NO:1 or NO:21, which ischaracterized by using Neuromedin U, a derivative thereof or a saltthereof and a protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.
 2. A kit for screening a compound or a salt thereofthat alters the binding property of Neuromedin U or a salt thereof witha protein or a salt thereof comprising the same or substantially thesame amino acid sequence as the sequence shown by SEQ ID NO:1 or NO:21,which is characterized by comprising Neuromedin U, a derivative thereofor a salt thereof and a protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.
 3. A compound or a salt thereof that alters thebinding property of Neuromedin U or a salt thereof with a protein or asalt thereof comprising the same or substantially the same amino acidsequence as the sequence shown by SEQ ID NO:1 or NO:21, which isobtainable using the screening method according to claim 1 or thescreening kit according to claim
 2. 4. A pharmaceutical compositioncomprising the compound according to claim
 3. 5. A pharmaceuticalcomposition according to claim 4, which is a therapeutic andprophylactic agent for obesity, hypertension or stress-related diseases.6. A screening method according to claim 1 or a screening kit accordingto claim 2, where Neuromedin U is a peptide comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:11.
 7. A protein or a salt thereof comprising the same orsubstantially the same amino acid sequence as the sequence shown by SEQID NO:1 or NO:21.
 8. A DNA containing the DNA encoding the proteinaccording to claim
 7. 9. A DNA according to claim 8 having the basesequence shown by SEQ ID NO:2 or No:22.
 10. A recombinant vectorcontaining the DNA according to claim
 8. 11. A transformant transformedwith the recombinant vector according to claim
 10. 12. A method forproducing the protein or salt thereof according to claim 7,characterized by cultivating the transformant according to claim 11 andmaking the transformant produce the protein according to claim
 7. 13. Anantibody to the protein or salt thereof according to claim 7.